scholarly journals Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Eric B. Miller ◽  
Sarah J. Karlen ◽  
Kaitryn E. Ronning ◽  
Marie E. Burns

Abstract Background The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging. Methods We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1–GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1–Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo. Results Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration. Conclusions The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1–Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

Author(s):  
Marco F. Taddio ◽  
Claudia A. Castro Jaramillo ◽  
Peter Runge ◽  
Alain Blanc ◽  
Claudia Keller ◽  
...  

Abstract Purpose The co-stimulatory molecules CD80 and CD86 are upregulated on activated antigen-presenting cells (APC). We investigated whether local APC activation, induced by subcutaneous (s.c.) inoculation of lipopolysaccharides (LPS), can be imaged by positron emission tomography (PET) with CD80/CD86-targeting 64Cu-labelled abatacept. Procedures Mice were inoculated s.c. with extracellular-matrix gel containing either LPS or vehicle (PBS). Immune cell populations were analysed by flow cytometry and marker expression by RT-qPCR. 64Cu-NODAGA-abatacept distribution was analysed using PET/CT and ex vivo biodistribution. Results The number of CD80+ and CD86+ immune cells at the LPS inoculation site significantly increased a few days after inoculation. CD68 and CD86 expression were higher at the LPS than the PBS inoculation site, and CD80 was only increased at the LPS inoculation site. CTLA-4 was highest 10 days after LPS inoculation, when CD80/CD86 decreased again. A few days after inoculation, 64Cu-NODAGA-abatacept distribution to the inoculation site was significantly higher for LPS than PBS (4.2-fold). Co-administration of unlabelled abatacept or human immunoglobulin reduced tracer uptake. The latter reduced the number of CD86+ immune cells at the LPS inoculation site. Conclusions CD80 and CD86 are upregulated in an LPS-induced local inflammation, indicating invasion of activated APCs. 64Cu-NODAGA-abatacept PET allowed following APC activation over time.


2021 ◽  
Vol 12 ◽  
Author(s):  
Yan Dong ◽  
Zhuo Wan ◽  
Xiaotong Gao ◽  
Guodong Yang ◽  
Li Liu

Cancer is one of the leading causes of death and a major public health problem all over the world. Immunotherapy is becoming a revolutionary clinical management for various cancer types. Restoration of aberrant immune surveillance on cancers has achieved markable progress in the past years by either in vivo or ex vivo engineering of the immune cells. Here, we summarized the central roles of immune cells in tumor progression and regression, and the existing and emerging strategies for different immune cell-based immunotherapies. In addition, the current challenges and the potential solutions in translating the immunotherapies into the clinic are also discussed.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2996-2996
Author(s):  
Gullu Gorgun ◽  
Teru Hideshima ◽  
Naoya Mimura ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
...  

Abstract Abstract 2996 Histone deacetylase 6 (HDAC6), a cytoplasmic enzyme, is a member of Class IIb HDAC family and catalyses the deacetylation of histones. Acetylation of histones induces chromatin relaxation leading gene transcription; conversely, deacetylation of histones induces chromatin condensation and silencing of gene transcription. HDAC6 regulates important cellular functions including histone modifications, gene transcription, and viral infection, degradation of misfolded proteins, cell migration, and immune synapse formation. HDAC inhibitors have been developed over the last decade as anti-tumor therapeutic agents in solid tumors and hematologic malignancies including multiple myeloma (MM). Myeloma represents a model system for the development of novel drugs which impact tumor cells as well as accessory cells in the tumor microenvironment. In this study we evaluated the effects of selective HDAC6 inhibition on immune accessory cells using a novel HDAC6 small molecule inhibitor, WT-161 compared with a control HDAC inhibitor trichostatin-A (TsA). Peripheral blood (PBMC) or bone marrow mononuclear cells (BMNC) from healthy donors or MM patients were cultured in the absence or presence of WT-161 (0.25-2.5uM) for 6h to 7 days. We first determined whether WT-161 induces HDAC6 inhibition in normal PBMCs by western blot of acetylated (Lys40)-tubulin, a target for HDAC6 activity. WT-161 induced acetylation of tubulin in PBMCs as early as 6 hours of exposure. Immunophenotypic changes were determined by flow cytometric analysis. Immunophenotypic analysis of PBMCs cultured with WT-161 showed significantly increased expression of PD1 in all immune cell subpopulations including CD4T, CD8T, CD3/CD8/CD56+ NKT and NK cells as well as in CD14+ myeloid cells. Conversely, decreased expression of negative costimulatory molecules CTLA4 and PDL1 was also noted, with no significant change in the expression of positive costimulatory molecules CD28, ICOS and ICOSL. Additionally, culture of PBMCs with WT-161 significantly increased expression of effector/memory T cell marker CXCR3 and decreased expression of CXCR4 in CD4T, CD8T, NKT, NK effector cells as well as CD14+ myeloid cells. WT-161 also induced ICAM1 expression on all immune cell subpopulations. WT-161 effects on the proliferation of immune cells were next assessed by CFSE-flow cytometric analysis. CFSE stained PBMCs were stimulated with anti-CD3 ab and cultured with WT-161 for 7 days. While WT-161 triggered proliferation of CD4T and CD8T cells, proliferation of NK and NKT cells decreased and B cells were unaffected. Interestingly, CD4/CD25/Foxp3 coexpressing regulatory T cells were also significantly diminished in both PBMCs and BMNCs cultured with WT-161 for 4 days. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in the immune effector cells in MM microenvironment. Ongoing analysis of effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies. Disclosures: Raje: AMGEN: Consultancy; Celgene: Consultancy; Novartis: Consultancy; astraZeneca: Research Funding. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson&Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.


1996 ◽  
Vol 7 (13) ◽  
pp. 1545-1553 ◽  
Author(s):  
James J. Mulé ◽  
Mary Custer ◽  
Bruce Averbook ◽  
James C. Yang ◽  
Jeffrey S. Weber ◽  
...  

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aubin Michalon ◽  
Andreas Hagenbuch ◽  
Christian Huy ◽  
Evita Varela ◽  
Benoit Combaluzier ◽  
...  

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.


2021 ◽  
pp. jim-2021-001788
Author(s):  
Xiumei Liu ◽  
Xueming Wang ◽  
Xiaoling Zhang ◽  
Ai hua Cao

Tic disorders (TD) are childhood-onset neurological disorders. Immune system dysregulation has been postulated to play a role in TD, and its mechanisms likely involve dysfunctional neural-immune cross-talk, which ultimately leads to altered maturation of the brain pathways that control different TD clinical manifestations and behavioral and emotional damages. Clinical studies have demonstrated an association between TD and allergies and overactive immune responses at a systemic level. In this study, the Yale Global Tic Severity Scale was taken as a global measure of tic severity. Compared with the control group, the group of children with TD plus allergic diseases displayed significantly increased Yale total scores (p<0.05), which suggests that children with TD plus allergic diseases have heavier tic symptoms. Both motor and vocal tic scores are higher in the group of children with TD plus allergy compared with the control group. We counted immune cell subpopulations using FACS. T lymphocyte subset comparison of CD3, CD4, CD8, and CD4:CD8 expression ratios revealed that the level of CD3, CD4, and CD4:CD8 in children with TD plus allergic diseases was significantly lower than those of children with TD without allergic diseases. These differences were statistically significant (p<0.05) and suggest that children with TD plus allergic diseases have imbalanced T lymphocyte subsets. We concluded that allergy increased the severity of TD through an imbalance in cellular immunity. Studies need to be done to show whether treatment of allergic symptoms leads to a decrease in TD manifestations.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroto Nakajima ◽  
Atsushi Miyashita ◽  
Hiroshi Hamamoto ◽  
Kazuhisa Sekimizu

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.


2018 ◽  
Author(s):  
Nicholas Borcherding ◽  
Kawther K. Ahmed ◽  
Andrew P. Voigt ◽  
Ajaykumar Vishwakarma ◽  
Ryan Kolb ◽  
...  

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.


eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marlies P Noz ◽  
Siroon Bekkering ◽  
Laszlo Groh ◽  
Tim MJ Nielen ◽  
Evert JP Lamfers ◽  
...  

Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.


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