Immune cell behaviour and dynamics in the kidney — insights from in vivo imaging

The advancement of new immunotherapies for the treatment of cancers, infections, immune-mediated inflammatory diseases, and autoimmune diseases necessitates the co-development of appropriate probes to detect and monitor the distribution and infiltration of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we have developed a set of novel single-domain antibodies (nanobodies, Nbs) that specifically recognize the human CD4 co-receptor in its native state on various CD4+ cells. Following detailed characterization of binding properties, epitope mapping, and site-directed functionalization, we selected biologically inert Nbs that do not affect T cell proliferation or cytokine expression in vitro. We used fluorescently labeled Nbs to track the presence and location of CD4+ cells in a xenograft model, demonstrating a high signal-to-background ratio by in vivo optical imaging. In summary, this study reports for the first time the generation and application of human CD4-specific Nbs for the detection and in vivo imaging of CD4+ cells in a preclinical animal model. We anticipate that the Nbs presented in this study will be versatile probes, e.g. in immunoPET imaging for patient stratification and for monitoring individual immune responses during personalized immunotherapy.

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Abstract WMP39: Indispensable Role of CCR5 Signaling In Tregs Protection Against Early Blood Brain Barrier Disruption After Stroke

Stroke ◽

10.1161/str.48.suppl_1.wmp39 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Peiying Li ◽

Long Wang ◽

Xiaoming Hu ◽

Jun Chen

Keyword(s):

In Vivo Imaging ◽

Immune Cell ◽

Immune Cell Infiltration ◽

Migration Test ◽

Ischemic Brain ◽

Two Photon ◽

Peripheral Immune Cell

Background and purpose: Our previous study found adoptively transferred regulatory T cell (Treg) protected against ischemic stroke via attenuating blood brain barrier (BBB) disruption. Here, we examined the role of CCR5, a chemokine receptor in the Tregs’ recruitment and protection to the ischemic BBB after stroke. Methods: Transient focal cerebral ischemia was induced in widetype C57/BL6 or CCR5(-/-) mice by unilateral middle cerebral artery occlusion (MCAO) for 60 minutes. Tregs (2x10 6 /mouse) in 200 μl PBS or PBS control were injected intravenously at 2 hours after MCAO. Chemo-attractant migration of Tregs to the ischemic BBB was examined by two photon in vivo imaging and cell migration test in vitro. RT-PCR, Immunofluorescence staining and confocal microscopy were used to assess the chemokine expression, endothelial ICAM-1 expression and peripheral immune cell infiltration following cerebral ischemic injury. In vivo endogenous IgG leakage and in vitro BBB integrity were examined to determine the important role of CCR5 in Tregs’ protection on the ischemic BBB. Results: We found that cerebral ischemia reduced Treg number in the peripheral but the expression of CCR5 on Tregs in the blood was increased. In the ischemic brain, the expression of CCR5 ligands were significantly upregulated, and the expression of CCL5 co-localized with ischemic brain endothelial cells. Genetic depletion of CCR5 on the donor Tregs significantly impaired the transferred Tregs’ ability to migrate to the ischemic BBB and attenuated Tregs’ protection against BBB disruption and peripheral immune cell infiltration as determined by two photon in vivo imaging, immunofluorescence staining and MMP-9 ELISA. In vitro migration test further revealed that depletion of CCR5 hindered Tregs to migrate across endothelial cell layer. Conclusions: In conclusion, CCR5 plays an indispensible role in Tregs’ recruitment and protection against ischemic BBB disruption. Therapeutic strategies targeting CCR5 warrants further investigation to improve Treg based cell therapy in stroke treatment.

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Faculty Opinions recommendation of In vivo imaging sheds light on immune cell migration and function in cancer.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727481941.793580083 ◽

2020 ◽

Author(s):

Paul Timpson

Keyword(s):

Cell Migration ◽

In Vivo Imaging ◽

Immune Cell ◽

Immune Cell Migration ◽

And Function

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In vivo Imaging of Tumor and Immune Cell Interactions in the Lung

BIO-PROTOCOL ◽

10.21769/bioprotoc.1973 ◽

2016 ◽

Vol 6 (20) ◽

Cited By ~ 2

Author(s):

Richard Hanna ◽

Grzegorz Chodaczek ◽

Catherine Hedrick

Keyword(s):

In Vivo Imaging ◽

Immune Cell ◽

Cell Interactions

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Fluorescent proteins for in vivo imaging, where's the biliverdin?

Biochemical Society Transactions ◽

10.1042/bst20200444 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2657-2667

Author(s):

Felipe Montecinos-Franjola ◽

John Y. Lin ◽

Erik A. Rodriguez

Keyword(s):

Hydrogen Peroxide ◽

In Vivo Imaging ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Imaging ◽

Infrared Light ◽

Red Fluorescent Protein ◽

Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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