Author response for "Nanoparticles containing constrained phospholipids deliver mRNA to liver immune cells in vivo without targeting ligands"

Background: Extrarenal 1α,25-dihydroxyvitamin D3 (1,25-D) locally produced by immune cells plays crucial roles in the regulation of the immune system. However, in vivo status of extrarenal 1,25-D and 25-hydroxyvitamin D (25-D) in acute inflammatory conditions are unknown. Objective: The aim of this study was to determine the extrarenal 1,25-D level in circulation in bilaterally nephrectomized rats, induced by low-dose lipopolysaccharide (LPS). Methods: Renal 1,25-D synthesis was terminated through bilateral nephrectomy in rats. The rats received intraperitoneal LPS (50 μg/kg BW) three times and the experiment was ended 24 hours after nephrectomy. Serum 1,25-D, 25-D, calcium, phosphorus, intact parathyroid hormone, and calcitonin levels were measured and immunohistochemistry was applied to detect the sources of extrarenal 1,25- D synthesis. Results: Circulatory 1,25-D concentration remarkably increased in both LPS-treated and non-treated bilaterally nephrectomized rats. Elevated circulatory 1,25-D did not have hypercalcemic endocrinal effects. The increased 1,25-D level also resulted in a concurrent rapid and dramatic depletion of circulatory 25-D. Conclusions: Extrarenal 1,25-D could enter into the systemic circulation and, therefore, might have systemic effects besides its autocrine and paracrine functions.

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A human antibody selective for transthyretin amyloid removes cardiac amyloid through phagocytic immune cells

Nature Communications ◽

10.1038/s41467-021-23274-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Aubin Michalon ◽

Andreas Hagenbuch ◽

Christian Huy ◽

Evita Varela ◽

Benoit Combaluzier ◽

...

Keyword(s):

Immune Cells ◽

Ex Vivo ◽

Phase 1 ◽

Hereditary Disease ◽

Elderly Subjects ◽

Human Antibody ◽

Cardiac Amyloid ◽

Healthy Elderly ◽

Ongoing Phase

AbstractTransthyretin amyloid (ATTR) cardiomyopathy is a debilitating disease leading to heart failure and death. It is characterized by the deposition of extracellular ATTR fibrils in the myocardium. Reducing myocardial ATTR load is a therapeutic goal anticipated to translate into restored cardiac function and improved patient survival. For this purpose, we developed the selective anti-ATTR antibody NI301A, a recombinant human monoclonal immunoglobulin G1. NI301A was cloned following comprehensive analyses of memory B cell repertoires derived from healthy elderly subjects. NI301A binds selectively with high affinity to the disease-associated ATTR aggregates of either wild-type or variant ATTR related to sporadic or hereditary disease, respectively. It does not bind physiological transthyretin. NI301A removes ATTR deposits ex vivo from patient-derived myocardium by macrophages, as well as in vivo from mice grafted with patient-derived ATTR fibrils in a dose- and time-dependent fashion. The biological activity of ATTR removal involves antibody-mediated activation of phagocytic immune cells including macrophages. These data support the evaluation of safety and tolerability of NI301A in an ongoing phase 1 clinical trial in patients with ATTR cardiomyopathy.

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FOXE1-Dependent Regulation of Macrophage Chemotaxis by Thyroid Cells In Vitro and In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms22147666 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7666

Author(s):

Sara C. Credendino ◽

Marta De Menna ◽

Irene Cantone ◽

Carmen Moccia ◽

Matteo Esposito ◽

...

Keyword(s):

Thyroid Cancer ◽

Immune Cells ◽

Cancer Susceptibility ◽

Macrophage Infiltration ◽

M2 Macrophage ◽

Thyroid Cells ◽

Transcriptional Alterations ◽

Cancer Phenotypes

Forkhead box E1 (FOXE1) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1-induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1. Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1-expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1. These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.

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102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0102 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A113-A113

Author(s):

Mireia Bachiller García ◽

Lorena Pérez-Amill ◽

Anthony Battram ◽

Alvaro Urbano-Ispizua ◽

Beatriz Martín-Antonio

Keyword(s):

T Cells ◽

Tumor Cell ◽

Cord Blood ◽

Immune Cells ◽

In Vivo Models ◽

Cell Clusters ◽

Cytotoxicity Assays ◽

Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

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620 Tumor cell-intrinsic STING pathway is activated in the presence of cues from immune cells and contributes to the anti-tumor activity of tumor cell-targeted STING agonist antibody-drug conjugates

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0620 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A656-A656

Author(s):

Naniye Malli Cetinbas ◽

Travis Monnell ◽

Winnie Lee ◽

Kalli Catcott ◽

Chen-Ni Chin ◽

...

Keyword(s):

Tumor Cell ◽

Cancer Cell ◽

Immune Cells ◽

Antibody Drug Conjugates ◽

Drug Conjugates ◽

Primary Monocyte ◽

Sting Pathway ◽

Anti Tumor Activity ◽

Antibody Drug

BackgroundSTING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to allow systemic administration while stimulating the innate immunity in a targeted manner. We have previously demonstrated that targeted delivery of a STING agonist with an ADC induces robust anti-tumor immune responses.MethodsHerein we investigated the mechanism of action of tumor cell-targeted STING agonist ADCs. We evaluated STING pathway activation and anti-tumor activity elicited by ADCs harboring either wild type (wt) or mutant Fc deficient in Fcγ receptor (FcγR) binding in wt or STING knockout (ko) cancer cell mono-cultures, immune cell co-cultures, and in in vivo tumor models.ResultsConsistent with previous reports, the majority of cancer cell lines tested failed to induce STING pathway following STING agonist payload treatment in mono-cultures. In cancer cell:THP1 monocytic cell co-cultures, tumor-targeted STING agonist ADCs with wt Fc exhibited robust STING activation, whereas Fc-mutant ADCs or non-targeted control ADCs had minimal activity. Similar results were obtained when THP1 cells were treated in plates coated with target antigen without cancer cells, demonstrating STING activation in THP1 cells following FcγR-mediated uptake of antigen-bound ADCs. Tumor-targeted Fc-wt ADCs led to marked induction of STING pathway and cancer cell-killing in cancer cell:PBMC or primary monocyte co-cultures, and complete tumor regressions in in vivo tumors. Surprisingly, while at reduced levels relative to the Fc-wt ADCs, Fc-mutant ADCs exhibited significant activity in these in vitro and in vivo models, suggesting that tumor cell-intrinsic STING pathway may be activated in the presence of cues from immune cells. Consistently, STING agonist payload treatment in the presence of conditioned media from PBMC and primary monocyte but not from THP1 cultures, led to STING activation in cancer cell mono-cultures. Moreover, Fc-mutant ADCs had diminished activity in STING ko cancer cell:PBMC or primary monocyte co-cultures, demonstrating the contribution of tumor cell-intrinsic STING activation to the anti-tumor activity elicited by tumor cell-targeted STING agonist ADCs.ConclusionsIn conclusion, we demonstrated that tumor cell-targeted STING agonist ADCs induce robust anti-tumor activity through mechanisms involving both FcγR and tumor antigen-mediated ADC internalization and subsequent induction of STING pathway in immune cells and tumor cells.

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Resolvin D1 reduces cancer growth stimulating a protective neutrophil-dependent recruitment of anti-tumor monocytes

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01937-3 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Domenico Mattoscio ◽

Elisa Isopi ◽

Alessia Lamolinara ◽

Sara Patruno ◽

Alessandro Medda ◽

...

Keyword(s):

Cancer Cells ◽

Growth Curve ◽

Immune Cells ◽

Cancer Growth ◽

Resolvin D1 ◽

Anti Cancer ◽

Classical Monocytes ◽

Human And Mouse

Abstract Background Innovative therapies to target tumor-associated neutrophils (PMN) are of clinical interest, since these cells are centrally involved in cancer inflammation and tumor progression. Resolvin D1 (RvD1) is a lipid autacoid that promotes resolution of inflammation by regulating the activity of distinct immune and non-immune cells. Here, using human papilloma virus (HPV) tumorigenesis as a model, we investigated whether RvD1 modulates PMN to reduce tumor progression. Methods Growth-curve assays with multiple cell lines and in vivo grafting of two distinct HPV-positive cells in syngeneic mice were used to determine if RvD1 reduced cancer growth. To investigate if and how RvD1 modulates PMN activities, RNA sequencing and multiplex cytokine ELISA of human PMN in co-culture with HPV-positive cells, coupled with pharmacological depletion of PMN in vivo, were performed. The mouse intratumoral immune cell composition was evaluated through FACS analysis. Growth-curve assays and in vivo pharmacological depletion were used to evaluate anti-tumor activities of human and mouse monocytes, respectively. Bioinformatic analysis of The Cancer Genome Atlas (TCGA) database was exploited to validate experimental findings in patients. Results RvD1 decreased in vitro and in vivo proliferation of human and mouse HPV-positive cancer cells through stimulation of PMN anti-tumor activities. In addition, RvD1 stimulated a PMN-dependent recruitment of classical monocytes as key determinant to reduce tumor growth in vivo. In human in vitro systems, exposure of PMN to RvD1 increased the production of the monocyte chemoattractant protein-1 (MCP-1), and enhanced transmigration of classical monocytes, with potent anti-tumor actions, toward HPV-positive cancer cells. Consistently, mining of immune cells infiltration levels in cervical cancer patients from the TCGA database evidenced an enhanced immune reaction and better clinical outcomes in patients with higher intratumoral monocytes as compared to patients with higher PMN infiltration. Conclusions RvD1 reduces cancer growth by activating PMN anti-cancer activities and encouraging a protective PMN-dependent recruitment of anti-tumor monocytes. These findings demonstrate efficacy of RvD1 as an innovative therapeutic able to stimulate PMN reprogramming to an anti-cancer phenotype that restrains tumor growth.

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A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

Scientific Reports ◽

10.1038/s41598-021-89882-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroto Nakajima ◽

Atsushi Miyashita ◽

Hiroshi Hamamoto ◽

Kazuhisa Sekimizu

Keyword(s):

Inflammatory Cytokines ◽

Immune Cells ◽

Ex Vivo ◽

Large Bubble ◽

Suitable Model ◽

Bacterial Cells ◽

Functional Impairments ◽

Gene Expressions ◽

Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

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Nanoparticles containing constrained phospholipids deliver mRNA to liver immune cells in vivo without targeting ligands

Bioengineering & Translational Medicine ◽

10.1002/btm2.10161 ◽

2020 ◽

Vol 5 (3) ◽

Cited By ~ 2

Author(s):

Zubao Gan ◽

Melissa P. Lokugamage ◽

Marine Z. C. Hatit ◽

David Loughrey ◽

Kalina Paunovska ◽

...

Keyword(s):

Immune Cells

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