IMAGING IMMUNITY – from Nanoscale to Macroscale | Insights from Biophysics
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POSTER SESSION II - also presented as Talks selected from Abstract Submissions

Session chair: Marleen Keyaerts (Brussels, Belgium); Rosa M. Moresco (Milano, Italy); Manfred Kneiling (Tübingen, Germany)
 
Date: Thursday, 16 January, 2020, 5:00 PM - 6:30 PM

To enhance discussions all talks selected from abstract submissions are also presented as posters. The best overall presentation (oral + poster) will receive an award!

Contents

Click on an contribution to preview the abstract content.

T-05

Can we image immune system activity using MRSI? Unraveling the oscillatory pattern of response in preclinical Glioblastoma (#1)

Shuang Wu1, Pilar Calero-Pérez1, Lucía Villamañan1, Nuria Arias-Ramos1, 2, Martí Pumarola3, 2, Sandra Ortega-Martorell4, Margarida Julià-Sapé2, 1, 5, Ana Paula Candiota2, 5, Carles Arús1, 2, 5

1 Universitat Autònoma de Barcelona, Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Cerdanyola del Vallès, Spain
2 CIBER Centro de Investigación Biomédica en Red- UAB, Cerdanyola del Vallès, Spain
3 Universitat Autònoma de Barcelona, Unit of Murine and Comparative Pathology.Department of Animal Medicine and Animal Surgery, Cerdanyola del Vallès, Spain
4 Liverpool John Moores University, Department of Applied Mathematics, Liverpool, United Kingdom
5 Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (IBB), Cerdanyola del Vallès, Spain

Introduction

Glioblastomas (GB) are malignant brain tumours with poor prognosis even after aggressive therapy (Temozolomide, TMZ, plus radiotherapy). We used volumetric MRSI-based nosological images (1) for GB therapy response assessment through tumour responding index (TRI) calculation. An oscillatory TRI pattern (6-7 days) was shown in longitudinal studies. The purpose of this work was to confirm TRI oscillations with Immune-Enhanced Metronomic (IMS) TMZ administration (2) and to characterize cellular populations contributing to MRSI spectral pattern changes in TMZ-treated preclinical GL261 GB.

Methods

GL261 GB tumours were induced in C57BL/6 mice (n=19) and TMZ administered every 6 days, at 60 mg/kg (n=13).  High resolution T2w MRI and consecutive 14 ms TE MRSI with 3-4 grids were acquired every 2 days (1) and nosologic maps calculated (3). Six mice selected by nosological images guided time points were euthanized for in vitro evaluation. Immunostainings for CD3 and Iba-1 were performed in TMZ-treated and control cases (n=4 and 2, respectively). Cured mice were followed-up by T2w MRI every 2 days and in case of no tumour mass detected after one month, a “rechallenge” experiment with new GL261 cell injection was carried out, contralateral to the initial injection site, to check out for anti-tumour immune memory, while wt mice (n=3) were also implanted in contralateral brain as controls.

Results/Discussion

IMS-TMZ treatment strongly increased survival for GL261 GB bearing mice, even producing cure (Fig. 1). Treated animals survived 210.7±174.1 days, improving previous results with a different TMZ administration protocol (33.8±8.7 days [3]), while untreated mice survived 21±1.5 days. TRI oscillations (6.2±1.5 days, Fig. 2A) were confirmed in IMS-TMZ treated mice, in agreement with 6.3±1.3 days reported in (1). MRSI spectral changes could reflect immune system presence/action, since macrophages can represent up to 30% of GB tumour mass (4). This is supported by immunohistochemistry results: CD3 was significantly different in responding and control zones (4.8±2.9 vs 3.3±2.5 positive cells/field, Fig. 2B). Iba-1 also showed significant differences (21.9±11.4 vs 16.8±9.7% of positive immunostained areas, Fig. 2C). Regarding the re-challenged mice, only one tumour grew after 10 days, and it was treated with IMS-TMZ, disappearing after one cycle. Tumours in wt control mice grew normally.

Conclusions

Our results indicate that host immune system is recruited against GL261 GB, and this can be imaged non-invasively by MRSI. Accordingly, this approach can be of interest for monitoring any type of therapy in which immune system participation is foreseen. Additionally, IMS-TMZ alone induced immune memory in immunocompetent C57BL/6 GB cured mice, as initially described (5), although additional work will be needed to clarify the ongoing mechanism.

References

1. Arias-Ramos, N, et al. Metabolites (2017) 7: pii: E20.
2. Ferrer-Font, L, et al. Pharmaceuticals (Basel) (2017) 10: pii:E24.
3. Delgado-Goñi, T, et al. NMR Biomed (2016) 29: 732-43.
4. Glass, R and Synowitz, M. Acta Neuropathol (2014) 128: 347-62.
5. Wu, J and Waxman, DJ. Oncoimmunology (2015) 4: e1005521.

Acknowledgement

This work was funded by the Ministerio de Economía y Competitividad (MINECO) grant MOLIMAGLIO (SAF2014-52332-R) to CA. Also funded by Centro de Investigación Biomédica en Red – Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, [http://www.ciber-bbn.es/en]), an initiative of the Instituto de Salud Carlos III (Spain) co-funded by EU Fondo Europeo de Desarrollo Regional (FEDER). APC received funding from the ATTRACT project funded by the EC under Grant Agreement 777222. We acknowledge the UAB Predoctoral training programme (PIF predoctoral fellowships for Ms P. Calero and L. Villamañan), Ministerio de Economia y Competitividad (FPI fellowship for Dr. N. Arias-Ramos) and also China Scholarship Council (predoctoral fellowship for Ms S. Wu).
Time allocation for MRI/MRSI acqusitions in Unit 25 of NANBIOSIS (www.nanbiosis.es) is gratefully acknowledged.

Treatment with IMS-TMZ can cure GL261 GB bearing mice

Figure 1. A) Kaplan Meier survival of GL261 tumour-bearing mice treated with vehicle (n=6) and TMZ (n=13) in an IMS protocol. B) Tumour volume evolution of cured mice group C) Graphical representation of tumour volume evolution, TRI oscillation and re-challenge point of  a cured mouse (C1276). Tumour reached maximum volume at day 17 p.i., then it was ablated after 7 doses of IMS-TMZ therapy, and the remaining scar was stable for one month. On day 74 p.i the mouse was re-implanted on contralateral parenchyma with GL261 cells. No tumour mass was detected within 3 months after re-injection.

Nosological image classification correlates with immune system elements

Figure 2: A) Top: tumour volume (mm3, black line) and % of responding pixels (% TRI, green line) for case C1264. Green columns indicate TMZ administration. Bottom: nosological images superimposed to MRI. Color-coding: Blue, normal; Red, non-responding tumour; Green, responding tumour. Black arrows indicate TMZ administration. Boxplot of B) CD3+ positive cells (n=147) and C) % of Iba-1 positive areas (n=148) in red and green areas of all studied cases. D) CD3+ and E) Iba-1 immunostaining for case C971. Nosological images superimposed to MRI. Arrows point to positive cells. Magnification (40×).

Keywords: MRI, MRSI, machine learning, brain cancer, response biomarker
T-02

Boiling Histotripsy Increases Systemic Immune Response against Murine Neuroblastoma Tumor and leads to Long-Term Survival (#9)

Avinash Eranki1, Mario Ries2, Priya Srinivasan1, Anthony Sandler1, AeRang Kim1, Karun Sharma1, Peter Kim7, Bradford Wood8, Chrit Moonen9

1 Children’s National Medical Center, The Sheikh Zayed Institute for Pediatric Surgical Innovation, Washington, United States of America
2 University Medical Center, Center for Imaging Sciences, Utrecht, Netherlands
3 The George Washington University, Department of Bioengineering and Surgery, Washington, United States of America
4 National Institutes of Health, Center for Interventional Oncology, National Cancer Institute, Bethesda, United States of America
5 University Medical Center, Center for Imaging Sciences, Utrecht, Netherlands

Introduction

Neuroblastoma (NBL) is a solid tumor that arises from the developing sympathetic nervous system and represents the most common solid childhood cancer with survival rates of only 20-40%. We previously demonstrated that high intensity focused ultrasound (HIFU) -mediated boiling histotripsy (BH) canproduce controlled, spatially precise mechanical tissue fractionation. The goal of this study was to evaluate the effect of BH to immunotherapy on improving survival in mice.

Methods

Mouse NBL tumors (Neuro2a) were grown in A/J mice via subcutaneous hind limb injection of 1×106 tumor cells. Tumors of 13-15 mm were treated with 1) BH alone (day 8), 2) ɑ-CTLA-4 + ɑ-PD-L1 (day 9, 12 and 15), or 3) BH + ɑ-CTLA-4 + ɑ-PD-L1 (N=10 each group). Mice were anesthetized and positioned using a custom-built holder and a 3-axis linear stage, and three foci (1.5×1.5×6 mm @-6dB) were treated within each tumor (fc=1.5 MHz, PRF=1 Hz, total sonication time/focus=15 s, and pulse length = 13 ms). Pain medication was administered in all mice. ELISA of cardiac blood serum, and flow cytometry of splenocytes were performed at three time points (Figure1a). Tumor sections were stained with H&E, CD4+ (helper T-cells), and CD68+ (macrophages) antibodies.

Results/Discussion

BH + ɑ-CTLA-4 + ɑ-PD-L1 significantly increased survival (62.5%), compared to BH alone or ɑ-CTLA-4 + ɑ-PD-L1 only (0% for both groups), with no recurrence (Figure1). BH resulted in ~40% tumor necrosis at 24-hrs (Figure2a,d). There was a significant increase in CD4+ and CD68+ population 24-hrs post BH (Figure2b,e, Figure2c,f, respectively). In addition, BH significantly reduced concentration of interleukin-10 (IL-10) at 24, 48, & 72-hrs, reflecting a significant increase in macrophages (Figure 2f). In addition, there was a significant increase in IL-2 at 24-hrs, strongly mirroring natural killer cells (NK-cells) activity presenting a similar trend. Interferon-gamma (IFN-ɣ) significantly increases at 72-hrs. Regulatory T-cells (FoxP3+) in the spleen significantly decrease by 72-hrs, and the ratio of CD8+/FoxP3+ significantly increases at 72-hrs post BH. All these results suggest that BH converts the NBL tumor from non-immunogenic to highly immunogenic.

Conclusions

We demonstrated that BH + ɑ-CTLA-4 + ɑ-PD-L1 is capable of treating large established NBL tumors in mice, resulting in long-term survival. BH increased CD8+ populations while reducing FoxP3+ populations and other immune suppressive cytokines, making the tumor more immunogenic. Our approach of using BH + checkpoint inhibitors, and demonstrating BH on a clinical HIFU system, potentially allows for rapid clinical translation of BH therapy (1).

References

1) Eranki et al. Clin. Cancer Res. in press

Figure 1

Kaplan-Meier plots of the three study groups. BH + ɑ-CTLA-4 + ɑ-PD-L1 group had significantly higher survival (62.5%) at 100 days compared to other groups.

Figure 2
Effects of BH on mouse neuroblastoma tumor. In comparison to untreated tissue (2a,b,c), BH caused tissue necrosis as confirmed on H&E (2d), and resulted in substantial increase in CD4+ and CD68+ populations within the tumor at 24-hrs (2e & 2f, respectively).
Keywords: Focussed Ultrasound, Histotripsy, Immune System Stimulation, Checkpoint Inhibition Therapy
T-01

Multimodal imaging identifies inflammation and fibrosis in response to pressure overload-induced heart failure and detects alleviation of cardiac remodeling following ventricular unloading (#15)

Aylina Glasenapp1, 2, Katja Derlin2, Gutberlet Marcel2, Laura B. N. Langer1, Hans-Jürgen Wester3, Tobias Ross1, Frank M. Bengel1, James T. Thackeray1

1 Hannover Medical School, Department of Nuclear Medicine , Hannover, Lower Saxony, Germany
2 Hannover Medical School, Department of Radiology, Hannover, Lower Saxony, Germany
3 Technical University of Munich, Radiopharmaceutical Chemistry, Garching, Bavaria, Germany

Introduction

Inflammation plays a crucial role in the progression of ischemic heart failure and contributes to fibroblast activation and scar formation. But the role of inflammation in non-ischemic heart failure is less well characterized. We hypothesized that non-invasive multimodality imaging would reveal parallel development of inflammation and fibrosis in response to pressure overload and heart failure. Mechanical unloading of the ventricle would attenuate inflammation and lower interstitial fibrosis resulting in restored contractile function.

Methods

C57Bl/6 mice underwent transverse aortic constriction (TAC, n=41) or sham surgery (n=20). To model mechanical unloading, the aortic banding was removed at 3wk post-surgery (rTAC, n=10). Serial PET images using the chemokine receptor CXCR4 ligand 68Ga-pentixafor were acquired at 1 and 3wk after TAC and 1wk and 3wk after rTAC to quantify cardiac inflammation. Cardiac magnetic resonance (CMR) at identical timepoints measured left ventricle (LV) geometry, ejection fraction (EF) and diffuse interstitial fibrosis by T1 mapping. Autoradiography and immunohistochemistry validated regional 68Ga-pentixafor distribution and inflammatory cell infiltration. Interstitial and perivascular fibrosis were confirmed by picrosirius red histology.

Results/Discussion

TAC increased LV mass (131±20 vs 87±7mg, p<0.001) and lowered ejection fraction (EF, 44±12 vs 66±6%, p<0.001) compared to sham. Failing hearts exhibited diffuse CXCR4 PET signal at 1wk after TAC (% injected dose (ID)/g, 0.96±0.20 vs 0.74±0.25, p<0.001) which declined by 3wk. Early CXCR4 PET signal predicted late EF (r=-0.42, p=0.01). Autoradiography confirmed tracer uptake (p=0.006), with proportional increase of CD68+ macrophages (p<0.01). Macrophage content inversely correlated with LV function (r=-0.82, p<0.01), and corresponded to regions of interstitial fibrosis. CMR T1 mapping revealed prolonged relaxation time after TAC (1151±139 vs 1006±34ms, p=0.003), proportional to EF (r=0.76, p<0.001) and histologic fibrosis (r=0.69, p=0.006). Ventricle unloading led to rapid EF recovery (57±13 vs 44±15%, p=0.022). Both CXCR4 PET signal and T1 relaxation were decreased after rTAC (0.75±0.19 vs 0.96±0.19%ID/g, p=0.026; 1002±61 vs 1151±139ms, p=0.004).

Conclusions

Inflammation and fibrosis occur early after pressure overload heart failure, which can be measured by 68Ga-pentixafor PET and CMR T1 mapping. These findings underscore the crucial relationship between inflammation and fibrosis in the failing heart. Ventricular unloading alleviates both inflammation and fibrosis which can be detected by imaging, suggesting the possibility to monitor therapeutic intervention in heart failure.

Multimodal imaging of inflammation and fibrosis after pressure overload heart failure

Figure 1 (A) Representative short axis PET images of chemokine receptor CXCR4 inflammation with 68Ga-pentixafor in the left ventricle defined by 18F-FDG; T1 maps at corresponding time points display relaxation time. (B) Quantification displays mild elevation of CXCR4 expression in the left ventricle after transverse aortic constriction (TAC) which is alleviated via reverse TAC unloading. (C) Mean T1 relaxation time is elevated after TAC and normalized by reverse TAC unloading.

Keywords: PET, MRI, Preclinical imaging, Inflammation, Heart failure
T-03

Human PD-L1 Nanobody For Immuno-PET Imaging: Strategies for Site-specific Radiolabeling (#17)

Jessica Bridoux1, Katrijn Broos2, Maxine Crauwels1, 3, Quentin Lecocq2, Charlotte Martin6, Frederik Cleeren7, Steven Ballet6, Guy Bormans7, Geert Raes5, Karine Breckpot2, Serge Muyldermans3, Nick Devoogdt2, Vicky Caveliers4, Marleen Keyaerts4, Catarina Xavier1

1 Vrije Universiteit Brussel (VUB), In Vivo Cellular and Molecular Imaging (ICMI), Jette, Belgium
2 VUB, Laboratory of Molecular and Cellular Therapy (LMCT), Jette, Belgium
3 VUB, Cellular and Molecular Immunology (CMIM), Ixelles, Belgium
4 UZ Brussel, Nuclear Medicine Department,, Jette, Belgium
5 VIB Inflammation Research Center, Myeloid Cell Immunology Lab, Ghent, Belgium
6 VUB, Research group of Organic Chemistry (ORGC), Ixelles, Belgium
7 University of Leuven, Radiopharmaceutical Research, Department of Pharmacy and Pharmacology, Leuven, Belgium

Introduction

Immune checkpoints such as Programmed death-ligand 1 (PD-L1) limit the T-cell function, and tumor cells have developed this receptor to escape the anti-tumor immune response. Monoclonal antibody-based treatments have shown long-lasting responses, but only in a subset of patients. Therefore, there is a need to predict the response to treatments. This study aims to develop a Nanobody (Nb)-based probe to assess human PD-L1 (hPD-L1) expression using PET imaging. The Nb has been site-specifically modified for Gallium-68 (68Ga) or Fluorine-18 (18F) radiolabeling.

Methods

The hPD-L1 Nb with a sortag-motif at its C-terminus was site-specifically coupled to a bifunctional chelator or a tetrazine (Tz) via the Sortase A enzyme coupling reaction. Modified Nbs were purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC), characterized by Mass Spectrometry (ESI-Q-TOF), SDS-PAGE and Western Blot. NOTA-(hPD-L1) Nb was labeled with 68Ga, RESCA-(hPD-L1) Nb with [18F]AlF and Tz-(hPD-L1) Nb with [18F]F-PEG3-BCN. Radiochemical purity (RCP) was assayed by SEC and iTLC. In vivo tumor targeting of [68Ga]Ga-NOTA-(hPD-L1) Nb was assessed in xenografted-athymic nude mice bearing PD-L1 positive cells, or PD-L1 negative cells as a control. PET/CT imaging was performed with 68Ga-labeled NOTA-(hPD-L1) Nb 1h post-injection.

Results/Discussion

Site-specifically functionalized hPD-L1 Nbs with NOTA or RESCA were obtained with high purity (≥99%) in 52% and 59% yield (Figure 1). (hPD-L1)-Tz was obtained in 63% yield. Functionalization did not affect affinity.
Labelling of NOTA-(hPD-L1) with 68Ga was performed at room temperature (RT) for 10 min in a 80% decay corrected radiochemical yield (DC-RCY), ≥99% RCP and apparent molar specific activity of 85 GBq/μmol. Over 4 hours, the radiolabeled probe and metal complex were stable (≥99% RCP). In vivo tumor targeting studies revealed high tumor uptake of (3.66 ± 0.76) %IA/g organ, and no unspecific organ targeting, except in the kidneys and excretion to the bladder (route of excretion).
Labeling of RESCA-(hPD-L1) with [18F]AlF was performed at RT for 12 min at in a 29% DC-RCY and with a RCP ≥99%. Biodistribution in healthy animals showed higher bone uptake than for the 68Ga-labeled Nb.
Preliminary results with [18F]F-PEG3-BCN-Nb showed about 30% labeling and more tests will be performed

Conclusions

A site-specific functionalized (hPD-L1) Nb was obtained using Sortase enzyme approach, which could be radiolabeled with 68Ga or 18F. [68Ga]Ga-NOTA-(hPD-L1) Nb proved to specifically target the hPD-L1 receptor in vivo. Further studies will be performed with the 18F-labeled Nb after optimization of the labelling conditions. The best probe in terms of ease of production and in vivobehavior will be selected for clinical translation.

References

K. Broos et. al., Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent, Cancers 2019, 11(6), 872.

Acknowledgement

The authors would like to thank Cindy Peleman for animal handling and PET imaging.Research project funded by the EU H2020 MSCA-ITN-2015 program 675417 PET3D. This work was funded by a grant from the Scientific Fund W. GeptsUZ Brussel and FWO G066615N.Q. Lecocqis funded by FWO-SB 1S24218N. Marleen Keyaerts is a senior clinical investigator of the Research Foundation – Flanders. Research at ICMI-BEFY is supported by the Strategic Research Program (SRP) of the VUB Research Council.

Figure 1: Site-specific functionalization and radiolabeling of the hPD-L1 Nb
Sortase A mediated site-specific functionalization of the hPD-L1 Nb with the bifunctional chelators (GGGYK-NOTA or GGGYK-RESCA) or with a tetrazine, and radiolabeling with Gallium-68, [18F]AlF or [18F]F-PEG3-BCN respectively.
Figure 2: Biodistribution of [68Ga]Ga-NOTA-(hPD-L1) Nb in tumor bearing mice.
Biodistribution of [68Ga]Ga-NOTA-(hPD-L1) Nb in athymic nude mice bearing MEL624 hPD-L1 positive (hPD-L1POS) tumors or negative (hPD-L1POS) tumors as a control, showing high specific tumor uptake of (3.66 ± 0.76) %IA/g organ, and no unspecific organ targeting, except in the kidneys and excretion to the bladder (route of excretion). (N = 6 / group). 
Keywords: PET-CT, nanobody, PD-L1, gallium-68, fluorine-18
T-04

Functionalized perfluorocarbon nanoemulsions for sensitive fluorine-19 MRI immune cell detection in vivo (#21)

Eric Ahrens1, Chao Wang1, Dina Hingorani1, Fanny Chaplin2, Benjamin Leach1, Stephen Adams3

1 Uc San Diego, Radiology, La Jolla, California, United States of America
2 UC San Diego, Bioegineering, La Jolla, California, United States of America
3 UC San Diego, Pharmacology, La Jolla, California, United States of America

Introduction

Advances in cell immunotherapy against cancer has stimulated the need for imaging tools to determine cell biodistribution and survival post-transfer. 19F MRI enables background-free, quantitative hot-spot imaging of cell therapies.1 We describe next-generation perfluorocarbon nanoemulsion (NE) probes to detect cells with an order of magnitude sensitivity improvement. We use a two-pronged approach for boosting detection via (i) the incorporation of paramagnetic metal chelate into the NE fluorous phase and (ii) formulation of NE displaying cell-penetrating peptides to enhance cell uptake.

Methods

We synthesized fluorinated, metal-binding β-diketones conjugated to linear perfluoropolyether (PFPE) yielding “FETRIS” construct2 and blended this with unconjugated PFPE oil. As a co-surfactant, we synthesized a modified peptide from the transactivator of transcription (TAT) component of the HIV virus type-1; TAT residues 49-58 to facilitate endocytosis, and a fluorous anchor was covalently attached. Fluorous TAT and poloxamer surfactant was used to form NE with the blended PFPE oil. Metalation of NE occurred via the addition of Fe(III) into the aqueous buffer. Intracellular cell uptake of the probe ex vivo was studied in human chimeric antigen receptor (CAR) T cells. In vivo 19F MRI mouse studies using inoculated labeled CAR T cells were performed in a xenograft model of glioma at 11.7 T.

Results/Discussion

Additional of Fe(III) into NE decreases the 19F T1 to >10-fold via the intermolecular paramagnetic relaxation enhancement mechanism, with modest T2 broadening. Shortening T1 increases the 19F image sensitivity per time with repetitive signal averaging. By incorporating TAT a labeling efficiency ~1012 fluorine atoms per CAR T cell was achieved which is a >8-fold increase compared to NE without TAT. In vitro assays show that T cells are unaltered after NE labeling. The 19F MRI signal detected from TAT-labeled CAR T cells in mouse was >8 times higher than cells labeled with control NE (Figure 1).

Conclusions

Lymphocytes are challenging to label and detect with MRI due to their weak phagocytic properties and small size. Using multipronged improvements to NE formulation via incorporation of Fe-chelate and TAT peptide, one can significantly enhance cell labeling and imaging sensitivity. These same agents should be useful for tagging other weakly phagocytic cells such as stem and progenitor cells.

References

1. Chapelin F, Capitini CM, Ahrens ET. Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer. J Immunother Cancer. 2018;6:105.

2. Kislukhin AA, Xu H, Adams SR, et al. Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging. Nat Mater. 2016;15(6):662-668.

Acknowledgement

We thank Hongyan Xu and Deanne Lister for technical assistance. ETA was funded through National Institutes of Health grants R01-EB024015 and the California Institute for Regenerative Medicine grant LA1-C12-06919. ETA is founder, consultant, member of the advisory board, and shareholder of Celsense, Inc.

Figure 1.

Panel (a) displays 19F (hot-iron) and 1H (grayscale) from a slice in mouse with bilateral flank gliomas, where the left and right tumor (LT, RT) each received 107 CAR T cells labeled with PFC (control) or TAT-PFC nanoemulsions, respectively. A reference (REF) is also shown. MRI data were acquired using RARE sequences. A histogram of the 19F signal-to-noise ratio for voxels in the tumors is displayed (b). Comparison of apparent 19F atoms per tumor in vivo (N = 4) is displayed (c) showing ~8-fold sensitivity enhancement (* indicates p<0.001) for TAT-PFC nanoemulsions compared to control.

Keywords: MRI, fluorine-19, lymphocytes, peptide, chelate
T-06

Intravital microscopy of fever-range hyperthermia as supporting strategy for cancer immunotherapy (#26)

Bettina Weigelin1

1 University of Tübingen, Preclinical Imaging and Radiopharmacy, Tübingen, Germany

Introduction

Adoptive transfer of ex vivo activated tumor-specific cytotoxic T cells (CTL) is a promising strategy to increase anti-tumor immunity, but its capacity to control tumor growth is often insufficient. External application of heat in the fever-range (38 – 40 °C) has been shown to activate and enhance immune effector function in tumors. However, the rational design of hyperthermia application schemes to support immunotherapy is currently hampered by an incomplete mechanistic understanding of how both therapies synergize.

Methods

By using time-lapse microscopy of tumor cells embedded in 3D collagen matrices and optical reporters, we monitored structural damage induced by OVA-specific CTL to the cellular and nuclear membranes and DNA double-strand breaks in B16F10/OVA melanoma cells under hyperthermia treatment. To evaluate the efficacy of adoptive CTL transfer in combination with whole-body hyperthermia (WBH) in the context of the melanoma microenvironment, we used intravital multiphoton microscopy combined with an imaging window for longitudinal monitoring of CTL effector function. Following intradermal tumor injection and adoptive CTL transfer, we applied WBH of 39.5 °C for 1 or 2 h and repeated the treatment every other day for one week.

Results/Discussion

CTL-mediated damage was predominantly sub-lethal and followed by rapid recovery of the tumor cell. Enhanced killing was observed already after treatments of 38.5 °C for 1 h on two consecutive days, while repeated, prolonged exposure to 40.5°C affected CTL viability and, consequently, killing capacity. Quantification of single CTL contacts with tumor cells derived from 48 h time-lapse recordings revealed that fever-range hyperthermia stabilized CTL–tumor cell contacts and impaired recovery of melanoma cells from CTL-mediated damage. In vivo imaging directly after treatment revealed an immediate block of tumor cell proliferation and increased apoptosis rates. Time-lapse microscopy showed enhanced CTL killing activity while CTL-tumor cell interaction dynamics remained unchanged, ranging from long-lasting to highly dynamic contacts. The combination of ACT and WBH further induced the infiltration of phagocytic cells which was absent in tumors of mice treated with either therapy alone.

Conclusions

Thus, kinetic imaging and intravital microscopy were successfully applied to deepen the mechanistic understanding of immune cell function during fever-range WHB which forms the basis for improved, rationale design of combination therapies.

Keywords: intravital microscopy, adoptive t cell transfer, hyperthermia
T-07

Imaging microglia/macrophages in vivo from microscale to macroscale in a murine model of ischemic stroke (#14)

Violaine Hubert1, Ines Hristovska2, Szilvia Karpati3, Frederic Lerouge3, Maelle Monteil4, Emmanuel Brun5, Naura Chounlamountri2, Chantal Watrin2, Fabien Chauveau6, Marc Lecouvey4, Stephane Parola3, Olivier Pascual2, Marlène Wiart1

1 Université de Lyon, CarMeN Inserm U1060, Bron, France
2 Université de Lyon, Institut NeuroMyoGène CNRS UMR 5310, Lyon, France
3 Université de Lyon, Laboratoire de Chimie CNRS UMR 5182, Lyon, France
4 Université Paris 13, Laboratoire CSPBAT CNRS UMR 7244, Bobigny, France
5 Université Joseph Fourier, STROBE team Inserm U647, Grenoble, France
6 Université de Lyon, BIORAN Team, Lyon Neurosciences Research Center, CNRS UMR5292 Inserm U1028, Bron, France

Introduction

Tissue-resident microglia and infiltrated macrophages (M/M) are important mediators of tissue damage after ischemic stroke and thus represent therapeutic targets. In the present study, our aim was to investigate the potential of MRI coupled with the injection of a novel nanoparticle (NP), NanoGd [1], to image M/M phagocytic activity at the acute stage of ischemic stroke. To understand the biological substrates of NanoGd-induced MR signals, we performed two-photon intravital microscopy back-to-back with MRI in CX3CR1-GFP mice submitted to permanent middle cerebral artery occlusion (pMCAO).

Methods

To evaluate NanoGd M/M internalization, microglial primary cultures were incubated with NanoGd at [0-1.5 mM] during 24h and imaged with confocal microscopy. To evaluate NanoGd biodistribution and pharmacokinetic, dynamic MRI of the abdomen was performed in 4 mice before, during and after i.v. bolus injection of NanoGd at 2 mmol Gd/kg.

At day 0 (D0), 22 mice underwent pMCAO. Baseline MRI was performed at day 1 (D1). NanoGd was then administered to 16 of the 22 operated mice at 2 mmol Gd /kg (Group I). The 6 other operated mice did not receive NanoGd and served as controls (Group II). Three nonoperated mice (sham) received NanoGd at the same dose (Group III). A subgroup of 11 mice were imaged with intravital two-photon microscopy at D1 and D2. All mice had follow-up MRI at day 3 (D3).

Results/Discussion

Confocal microscopy of microglial cultures incubated with NanoGd showed an internalization by Iba-1+ cells (Fig 1A). Abdominal MRI showed a strong uptake of NanoGd in liver and spleen at 1h post-injection (Fig 1B) and a prolonged vascular remanence (blood half-life estimated > 6h). These data were used to design the in-vivo study (Fig 1C): post-NanoGd MRI was scheduled at 48h to allow time for NanoGd to be eliminated from the vascular bed at the time of follow-up MRI.

Baseline MRI showed blood brain barrier disruption in the ischemic lesion of all pMCAO mice (Fig 2A-B). Follow-up T2WI and T2*WI showed strong hypointense MR signals in the ischemic lesion of pMCAo mice, not found in control groups (Fig 2C-D). Intravital two-photon images of the same mice confirmed NanoGd extravasation in brain parenchyma and internalization by CX3CR1+ cells in the ischemic lesion but not in the extralesional area (Fig 2E-F). Post-mortem analyses of perfused brain corroborated these results (Fig 2E-F).

Conclusions

The present study builds upon our former works using NP-enhanced MRI to monitor M/M in vivo at the acute stage of stroke [2-4]. We confirm and extend our previous findings by performing the very first study using MRI and intravital bi-photon microscopy back-to-back in a murine model of ischemic stroke, thanks to a new multimodal NP. Our on-going work aims at investigating the immunomodulatory effects of simvastatin with this bimodal approach.

References

1.           Halttunen N, Lerouge F, Chaput F, Vandamme M, Karpati S, Si-Mohamed S, et al. Hybrid Nano-GdF3 contrast media allows pre-clinical in vivo element-specific K-edge imaging and quantification. Scientific reports. 2019;9(1):12090.

2.           Desestret V, Brisset JC, Moucharrafie S, Devillard E, Nataf S, Honnorat J, et al. Early-stage investigations of ultrasmall superparamagnetic iron oxide-induced signal change after permanent middle cerebral artery occlusion in mice. Stroke. 2009;40(5):1834-41.

3.           Marinescu M, Chauveau F, Durand A, Riou A, Cho TH, Dencausse A, et al. Monitoring therapeutic effects in experimental stroke by serial USPIO-enhanced MRI. Eur Radiol. 2013;23(1):37-47.

4.           Wiart M, Davoust N, Pialat JB, Desestret V, Moucharaffie S, Cho TH, et al. MRI monitoring of neuroinflammation in mouse focal ischemia. Stroke. 2007;38(1):131-7.

Acknowledgement

The authors thank Radu Bolbos and Jean-Baptiste Langlois of the Animage platform (CERMEP, Lyon) for their technical assistance. This research was funded by the French national research agency (ANR) project NanoBrain (grant # ANR-15-CE18-0026-01) and was performed within the framework of the RHU MARVELOUS (ANR16-RHUS-0009) of University Claude Bernard Lyon  (UCBL), within the program “Investissements d’Avenir”. We also thank ESRF for allocation of beamtime.

Figure 1- NanoGd characterization and study design.
A. Confocal images of Iba-1 stained microglial cells incubated without NanoGd (A1) or with NanoGd (A2, 0.5 mmol/L). Scale bars: 50 µm for overview images; 10 µm for magnified insets.  B. NanoGd biodistribution assessed by abdominal dynamic MRI. Left images were acquired before NanoGd injection and right images after NanoGd injection, at the end of the dynamic sequence (1h post-injection). Green arrows point out the hypointense signals induced by NanoGd in the organs of interest. C. In-vivo study design. pMCAO: permanent middle cerebral artery occlusion. 2γ µscopy: two-photon microscopy.
Figure 2- Spatiotemporal pattern of NanoGd distribution following pMCAo.

Pre- (A-B) and post-NanoGd (C-D) MRI: T2-WI shows the presence of an ischemic lesion in pMCAo mice (dotted white lines), but not in the sham mouse. Blood brain barrier breakdown was assessed using a T1-WI post-Gd (white arrowheads). Hypointense signals are observed with T2-WI (C) and T2*-WI (D) 48h post-NanoGd injection (red arrowheads). Scale bars: 1mm. (E-F) Representative images of two-photon microscopy sessions at D1 and D2 in the extralesional area (E) and ischemic core (F). Scale bar: 20µm. Post mortem examination, scale bars: 50 µm for overview images; 20 µm for magnified insets.

Keywords: MRI, intravital two-photon microscopy, pathophysiology, ischemic stroke, nanotechnology
T-08

Investigating the Cardiorenal Axis using CXCR4-directed Imaging (#11)

Rudolf A. Werner1, Annika Heß1, Tobias Koenig2, Johanna Diekmann1, Thorsten Derlin1, Hans-Jürgen Wester3, Anette Melk4, Johann Bauersachs2, James T. Thackeray1, Frank M. Bengel1

1 Hannover Medical School , Department of Nuclear Medicine, Hannover, Germany
2 Hannover Medical School , Department of Cardiology and Angiology, Hannover, Germany
3 Technical University Munich, Pharmaceutical Radiochemistry, Munich, Germany
4 Hannover Medical School , Department of Kidney, Liver and Metabolic Diseases, Children's Hospital, Hannover, Germany

Introduction

Cardiorenal syndrome comprises a spectrum of disorders involving bidirectional interaction between the failing heart and the kidney, wherein adaptive immune responses are a key pathophysiological mechanism. We aimed to obtain further insights into the cardiorenal crosstalk using systems-based whole-body chemokine receptor CXCR4-directed imaging to assess inflammation in the heart and kidney.

Methods

After permanent myocardial infarction (MI) or sham surgery, serial PET imaging with the CXCR4 specific ligand 68Ga-pentixafor was conducted in C57Bl6/N mice (n=80) at 1d, 3d, 7d, and 6wk after surgery. Contractile function was evaluated by cardiac magnetic resonance imaging. Additionally, 30 patients underwent CXCR4-directed PET imaging median 3.4 days after acute MI. Laboratory values of renal function (creatinine) were collected at baseline and after a median follow-up of 8 months.

Results/Discussion

MI mice demonstrated maximal cardiac CXCR4 upregulation at day 1 compared to sham, followed by a gradual decline (infarct/remote ratio: 1.40±0.12 at 1d*, 1.33±0.12 at 3d*, 1.11±0.07 at 7d*, 1.00±0.09 at 6wk, *P<0.05 vs. sham). Cardiac CXCR4 signal was paralleled by elevated splenic and renal CXCR4 signal (infarct/kidneys: 0.90±0.18 at d1*, 0.73±0.13 at 3d*, 0.49±0.14 at 7d, 0.43±0.09 at 6wk, *P<0.0001 vs. sham). Renal 68Ga-pentixafor uptake at 7d after MI in mice predicted left ventricular remodeling at 6wk (left ventricular ejection fraction, LVEF≤30%, R=-0.79, P<0.05 vs. LVEF>30%, R=-0.49, n.s.). Among MI patients, renal CXCR4 signal was independent of creatinine at baseline (R=-0.02, n.s.), but correlated with LVEF normalised to the infarct signal (R=-0.39, P<0.05). Early renal CXCR4 upregulation, however, showed a trend to correlate with late kidney function, which was more pronounced relative to the infarct signal (kidneys: R=0.42 vs. infarct: R=0.29, n.s., respectively).

Conclusions

Inflammatory crosstalk between failing heart and kidneys can be monitored non-invasively using CXCR4-directed PET imaging. Such systems-based whole-body imaging strategies may open avenues for further mechanistic insights into the cardiorenal axis and provide a foundation for future exploration of strategies pursuing imaging-guided multi-organ reparative therapies.

Acknowledgement

This work was funded by the German Research Foundation (DFG), through the Clinician-Scientist program PRACTIS (RAW) and the clinical research group KFO311 (FMB, JB). No potential conflicts of interests relevant to this article exist.

Investigating the cardiorenal axis using CXCR4-directed imaging in a translational approach.

Upper row: Serial short axis images of the myocardium and coronal images of the spleen and the kidneys in sham and after acute myocardial infarction (AMI) in mice (left). Renal CXCR4 signal predicted late left ventricular ejection fraction (LVEF) in mice at 7 days after MI (right). Lower row: In patients after AMI (left), renal CXCR4 signal predicted renal function derived by serum-creatinine during follow-up (right).

Keywords: PET, Nuclear Medicine, Molecular Imaging, Heart, Kidneys
T-09

Molecular imaging of chemokine receptor CXCR4 early after myocardial infarction to assess inflammation and to guide therapy (#8)

Annika Hess1, Alexander Wittneben1, Hans-Jürgen Wester2, Tobias Ross1, Frank M. Bengel1, James T. Thackeray1

1 Hannover Medical School, Department of Nuclear Medicine, Hannover, Lower Saxony, Germany
2 Technical University of Munich, Chair of Radiopharmaceutical Chemistry, Garching, Bavaria, Germany

Introduction

Myocardial infarction (MI) induces tissue inflammation, which requires balance between pro- and anti-inflammatory components for effective cardiac repair. Targeted anti-inflammatory therapies are emerging, but patient selection remains challenging due to heterogeneity. We hypothesized that quantitative PET imaging of chemokine receptor CXCR4 and leukocyte infiltration after MI could predict acute and chronic outcome and subsequently guide CXCR4-targeted therapy.

Methods

C57Bl6/N mice underwent permanent ligation (n=149) or 60 min ischemia/reperfusion (n=9) of the left coronary artery, or sham surgery (n=13). Myocardial inflammation was assessed in subgroups of mice by serial CXCR4 PET (n=126) and ex vivo autoradiography (n=4) with 68Ga-pentixafor over 1wk post-MI. Perfusion SPECT defined infarct sizes, and cardiac magnetic resonance assessed left ventricular (LV) function. Targeted anti-inflammatory treatment with the CXCR4 antagonist AMD3100 (125µg) was administered at 1h (n=13), 3d (n=24), or 7d (n=16) post-MI, based on the CXCR4 PET signal intensity. Immunohistochemistry (n=4) and flow assisted cell sorting (FACS, n=40) assessed leukocyte accumulation in the heart on each PET imaging timepoint.

Results/Discussion

Infarct CXCR4 PET signal was highest at 1h post-MI versus sham (1.3±0.2%ID/g vs 0.6±0.1, p<0.001) and declined by 7d (0.6±0.1, p=0.9), confirmed by FACS and histology. Splenic CXCR4 signal was increased and proportional to the infarct signal (d1: r=0.56, p=0.001; d3: r=0.65, p<0.001), reflecting leukocyte activation. Persistent infarct CXCR4 upregulation at 3d post-MI predicted acute LV rupture compared to survivors (d3: 1.2±0.3 vs 0.9±0.2%ID/g, p<0.001). Among survivors, CXCR4 signal at 3d independently predicted ejection fraction at 6wks (36±15 vs 70±4%, p=0.001; rpartial=-0.4, p=0.04). Following the PET timecourse, selective CXCR4 blockade at 1h or 3d post-MI markedly lowered incidence of acute LV rupture compared to untreated MI (0% vs 8% vs 22%) and improved 6wk EF (+34% (1h), +24% (3d), p=0.01). FACS analysis revealed lower leukocyte content after CXCR4 blockade on d3 (p<0.04). CXCR4 blockade at MI+7d, outside PET-defined upregulation, did not improve survival or function.

Conclusions

PET imaging in mice identifies dynamic CXCR4 upregulation over the first week post-MI, which predicts early survival and late cardiac function. Image-guided blockade of CXCR4 at precise timepoints accelerates inflammatory resolution and improves acute and chronic cardiac outcome. This work provides the foundation for PET-guided CXCR4-targeted therapy after MI which should be further evaluated in the clinical setting.

PET imaging to guide CXCR4-targeted anti-inflammatory therapy

Figure. (A) Longitudinal short axis cardiac 68Ga-pentixafor PET images display CXCR4 upregulation (colorscale) in infarct territory defined by 18F-FDG (greyscale) at 1h and 3d but not 7d after MI. A single injection of CXCR4 blocker AMD3100 at timepoints corresponding to PET-defined CXCR4 upregulation (1h or 3d) but not off-peak (7d) evokes (B) lower incidence of acute left ventricular (LV) rupture within the first 7d post-MI, and (C) significantly improves chronic LV ejection fraction at 6wks compared to untreated MI.

Keywords: PET, MRI, Myocardial inflammation, Anti-inflammatory therapy
T-10

Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model (#13)

Celine Caravagna1, 2, Alexandre Jaouen1, 2, Sophie Desplat-Jego4, Keith Fenrich1, Elise Bergot4, Hervé Luche3, Pierre Grenot3, Geneviève Rougon1, 2, Marie Malissen4, 3, Franck Debarbieux1, 2

1 Aix Marseille University - CNRS, Institut de Neurosciences Timone, Marseille, France
2 Aix Marseille University - CNRS, CERIMED, Marseille, France
3 Aix Marseille University - INSERM, CIPHE, Marseille, France
4 Aix Marseille University - CNRS-INSERM, CIML, Marseille, France

Introduction

In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage.

Methods

To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice

Results/Discussion

We have identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP+ neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP+/EYFP+ monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris.

Conclusions

LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.

References

  1. Jaouen A., Caravagna C., Desplat-Jego S., Fenrich K., Bergot E., Luche H., Grenot P., Rougon G., Malissen M., Debarbieux F. (2018) Diversity of innate immune cell subsets across spatial and temporal scales in a EAE mouse model. Scientific Reports 23;8(1):5146.
  2. Fenrich K.K., Weber P., Rougon G, Debarbieux F. (2013) Long and short term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury J Physiol. 591: 4895-4902

Acknowledgement

We thank Marion Compagnone and Florence Pelletier for mouse colony management. Drs Carole Colin and Jacques Durand for statitical analyses. This work was supported by Agence Nationale Recherche ANR15-CE16-0009-01 (to F.D., M.M.),  and Association de Recherche sur la Sclérose en Plaques ARSEP grants 2015 (to G.R., M.M.), a CIFRE fellowship (to A.J.), a Marie Skłodowska-Curie Actions fellowship from European Commission program H2020 (to C.C.), and core support from AMU, CNRS and INSERM

Characterization of the fluorescently labeled immune cells in Thy1-CFP//LysM-EGFP//CD11c-EYFP mice
A) LysM-EGFP and CD11c-EYFP expression in microglia and the various infiltrating immune cell  from EAE brain at day 17 B) Distribution histograms for microglia cells C)  Bar graph showing the expression of MHC class II, CD11c and CD11c-EYFP in microglia cells D) Quantification and distribution of fluorescent cells in the EAE spinal cord for control (PBS or CFA.PTX) or induced (MOG.CFA.PTX) mice at day 8, 13 or 17. The pie chart sizes are proportional to the total number of cells. The numbers indicate the percentage of the corresponding population
In vivo visualization of immune cell dynamics during EAE progression.
EGFP+ cells (green), EYFP+ cells (yellow), EGFP+/EYFP+ cells (pink, manually highlighted), CFP+ neurons (cyan), blood vessels (red). D = post-induction day. A) Dynamics of cell distribution during EAE at different post-induction days. B) Evolution of immune cell densities relative to pre-induction values  C)  Initial meningeal accumulation of EGFP+ cells and subsequent infiltration into the tissue. D)  Evolution of the number of CFP+ axons in determined regions of interest at different stages of EAE progression E) Evolution of identified individual axons F) Axonal damages
Keywords: MODALITY: Intravital 2P-fluorescence microscopy, DOMAIN: Neuroinflammation, preclinical imaging
T-11

In vivo tracking of polyclonal human regulatory T cells reveals a role for innate immune cells in Treg transplant recruitment. (#18)

Jacinta Jacob2, Yasmin Mohseni1, 2, Alessia Volpe1, Qi Peng2, Suchita Nadkarni3, Rosalind Hannen3, Robert I. Lechler2, Federica Marelli-Berg3, Lesley Smyth4, Giovanna Lombardi2, Gilbert O. Fruhwirth1

1 King's College London, Imaging Chemistry & Biology, Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 King's College London, MRC Centre for Transplantation, Peter Gorer Department of Immunobiology, School of Immunology and Microbial Science, London, United Kingdom
3 Queen Mary University London, William Harvey Research Institute, London, United Kingdom
4 University of East London, School of Health, Sport and Bioscience, London, United Kingdom

Introduction

In solid organ transplantation, organ demand outstrips supply with allograft rejection being another limitation. Adoptive transfer of regulatory T-cells (Tregs) protects from graft rejection1, and its safety has been shown in clinical trials. Antigen-specific Tregs are superior to polyclonal Tregs, with chimeric antigen receptors (CAR) emerging as an attractive option to produce them2. Important questions about in vivo fate, distribution and localization of Treg function remain unclear. Our goal was to identify a long-term in vivo tracking approach compatible with future clinical translation.

Methods

Human Tregs enriched via a GMP-compatible protocol were either directly labelled usgin 89Zr-oxine or lentivirally transduced with the reporter human sodium iodide symporter (NIS) fused to a fluorescent protein (FP) for preclinical evaluation3. Antigen-specific Tregs were produced using an expression platform for both a HLA-A2-specific CAR4 and the reporter. Tregs were characterized in vitro for phenotype, survival/expansion, suppressive capacity/activatability, and radiotracer (99mTcO4-) uptake if NIS-transduced. BALB/c Rag2‑/‑γc‑/‑ (BRG) mice were transplanted with A2+ human skin and ~6 weeks later traceable Tregs were i/v administered. Transplants were monitored by nanoSPECT/CT up to 40d for Treg recruitment/presence. Ex vivo tissue histology was employed to support in vivo data.

Results/Discussion

89Zr-labelling was unsuitable due to excessive cellular radiodamage affecting Treg survival/expansion at cellular label concentrations required for long-term in vivo Treg tracking (Fig.1). In contrast, reporter engineered Tregs fully retained phenotypes (CD4/CD25/FOXP3) and function (effector T-cell suppression, activatability/CD69) while showing stable reporter expression over weeks (Fig.2A-C). Antigen-specific CAR-Tregs showed significantly higher CD69 activation when challenged with A2+ B-cells compared to A2- B-cells (Fig.2D). Radiolabelling did not impact on Treg function (Fig.2E). NIS-FP+ Tregs were successfully tracked in vivo by SPECT/CT imaging (Fig.2F-G) and detectable from day 3 reaching a peak at day 8 and persisting in human skin grafts for at least 40 days. Importantly, we found early trafficking of Tregs to skin grafts to be markedly reduced by elimination of recipient innate Gr‑1+ immune cells (neutrophils and distinct monocyte subsets).

Conclusions

We showed first proof-of-principle of in vivo Treg tracking to transplants. This data suggests utility of radionuclide reporter gene imaging as a clinically compatible strategy for long-term in vivo Treg tracking in future clinical trials. Notably, with CAR-Treg therapy likely to lead the way, and thereby genetic engineering becoming a fundamental requirement, reporter gene technology does not add undue complications to this cell therapy.

References

1 Safinia N, et al. Cell Therapy in Organ Transplantation: Our Experience on the Clinical Translation of Regulatory T Cells. Frontiers in immunology 2018, 9:354.
2 Boardman DA et al. Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection. Am J Transplant 2017, 17(4):931.

3 Volpe A, Man F, Lim, L, Khoshnevisan A, Blower JE, Blower PJ, Fruhwirth GO. Radionuclide-fluorescence reporter gene imaging to track tumor progression in rodent tumor models. J Vis Exp 2018; 133:e57088. doi: 10.3791/57088.

Acknowledgement

We acknowledge funding from the Medical Research Council UK, the British Heart Foundation and Cancer Research UK. We declare no conflict of interest.

Figure 1
Figure 2
Keywords: Immunology, SPECT/CT, reporter gene, transplantation, regulatory T-cells
T-12

Tumor immune escape via tumor hypoxia and acidosis mediated PD-L1 expression as a prognostic biomarker (#30)

Philipp Knopf1, Natalie Mucha1, Andreas Maurer1, 12, Marilena Poxleitner1, Omelyan Trompak3, Bredi Tako1, Maren Harant1, Lars Zender3, 12, Marieke Fransen4, Irene Gonzalez-Menendez4, Balaji Krishnamachary7, Birgit Fehrenbacher2, Martin Schaller2, Daniela Kramer5, Leticia Quintanilla-Martinez4, 12, Klaus Schulze-Osthoff10, 11, 12, Zaver M. Bhujwalla7, 8, 9, Kamran Ghoreschi2, 6, Bernd Pichler1, 12, Manfred Kneilling1, 2, 12

1 Eberhard Karls University Tübingen , Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany
2 Eberhard Karls University Tübingen , Department of Dermatology, Tübingen, Baden-Württemberg, Germany
3 Eberhard Karls University Tübingen , Department of Internal Medicine VIII, Tübingen, Baden-Württemberg, Germany
4 Leiden University Medical Center , Department of Immunohematology and Blood Transfusion, Leiden, Netherlands
5 Eberhard Karls University, Institute of Pathology and Neuropathology, Tübingen, Baden-Württemberg, Germany
6 Charité – Universitätsmedizin Berlin, Department of Dermatology, Venereology and Allergology, Berlin, Berlin, Germany
7 The Johns Hopkins University, School of Medicine, Division of Cancer Imaging Research, Baltimore, Maryland, United States of America
8 The Johns Hopkins University, School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, United States of America
9 The Johns Hopkins University, School of Medicine, Department of Radiation Oncology and Molecular Radiation Sciences, Baltimore, Maryland, United States of America
10 Eberhard Karls University of Tübingen, Interfaculty Institute of Biochemistry, Tübingen, Baden-Württemberg, Germany
11 German Cancer Research Center, German Cancer Consortium (DKTK), Heidelberg, Baden-Württemberg, Germany
12 Cluster of Excellence iFIT (EXC 2180), "Image Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Tübingen, Baden-Württemberg, Germany

Introduction

Cancer and immune cells, depend on the presence of glucose and oxygen, whereas anaerobic glycolysis leads to lactate production and low pH values. Tumor acidosis inhibits T cell function and together with hypoxia represent a tumor escape mechanism by up-regulation of programmed death ligand-1 (PD-L1)1. Thus, targeting PD-L1/PD-1 axis by monoclonal antibodies (mAbs) is a successful treatment strategy. The aim of our study was to image tumor hypoxia in vivo by optical imaging during tumor pH neutralization and elucidate the underlying mechanisms of acidosis and IFN-γ mediated PD-L1 expression.

Methods

We s.c. injected murine 5HREp-ODD-luc transduced MC38 cells (MC38-HRE-ODD-Luc) in mice treated with sodium bicarbonate or normal drinking water to longitudinally monitor the presence of tumor hypoxia non-invasively in vivo with optical imaging. Furthermore, we illuminated the mechanism of acidosis and IFN-γ induced PD-L1 up-regulation in vitro in murine MC38 as well as human HCA-7 tumor cells using siRNA mediated STAT1 knockdowns, fluorescence microscopy, flow cytometry, western blot and qRT-PCR. In vivo extracellular tumor pH neutralization experiments by sodium bicarbonate treatment using tumor models that respond (MC38 and CT26) or not respond (B16-F10 and 4T1) to anti-PD-L1 mAbs therapy were performed and tumors were resected for histopathological analysis and CD3 immunohistochemistry.

Results/Discussion

In in vivo experiments we determined in sodium bicarbonate-treated MC38-HRE-ODD-Luc tumor bearing mice an elevated luciferase expression most likely as a consequence of an enhanced T cell recruitment and activation. In vitro, IFN-γ and acidic media caused an increased PD-L1 expression in murine MC38 cells. A similar IFN-γ and acidic media induced PD-L1 up-regulation was found in human HCA-7, MCF-7 and U-87 MG cells. In vitro STAT1 siRNA knockdown experiments revealed that IFN-γ and acidosis induced up-regulation of PD-L1 is mediated by pronounced STAT1 expression and activation in a temporally resolved manner. Single sodium bicarbonate as well as combined sodium bicarbonate & PD-L1 mAbs treatment of MC38 or CT26 tumor bearing mice yielded a reduction in tumor volume and a pronounced tumoral homing of T-cell when compared to sham-treated mice. In contrast, single sodium bicarbonate treatment or single anti-PD-L1 mAbs treatment was inefficient in B16-F10 and 4T1 tumor bearing littermates.

Conclusions

IFN-γ together with acidosis increases PD-L1 expression by increased STAT1 expression and activation. Most interestingly, PD-L1 mAbs responsive tumor models are additionally responsive to sodium bicarbonate treatment, while tumors not responsive to PD-L1 mAbs treatment are also not responsive to extracellular tumor pH neutralization treatment. Thus, IFN-γ and acidosis induced up-regulation of PD-L1 might explain a tumor resistance mechanism.

References

1              Huber, V. et al. Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation. Seminars in cancer biology 43, 74-89, doi:10.1016/j.semcancer.2017.03.001 (2017).

Acknowledgement

PK was supported by a grant from the German Academic Exchange Service (DAAD PPP USA 2018, Project-ID 57387312)

Keywords: PD-L1, Hypoxia, Acidosis, Checkpoint Inhibitor
T-13

A multimodal in vivo imaging approach to longitudinally assess and quantify infection and immune cell infiltration in preclinical models of fungal infections. (#3)

Shweta Saini1, Hannelie Korf3, Rein Verbeke5, James Dooley2, 4, Greetje Vande Velde1, Stefaan Soenen1, Conny Gysemans7, Katrien Lagrou6, Stefaan De Smedt5, Ine Lentacker5, Adrian Liston2, 4, Uwe Himmelreich1

1 University of Leuven, Biomedical MRI/ Dept Imaging and Pathology, Leuven, Belgium
2 The Babraham Institute, The Babraham Institute Cambridge, Cambridge, United Kingdom
3 University of Leuven, Laboratory of Hepatology, Leuven, Belgium
4 University of Leuven, Laboratory of Genetics of Autoimmunity, Leuven, Belgium
5 University of Ghent, Ghent Research Group on Nanomedicines, Ghent, Belgium
6 University of Leuven, Clinical Bacteriology and Mycology, Leuven, Belgium
7 University of Leuven, Clinical and Experimental Endocrinology, Leuven, Belgium

Introduction

Fungal infections caused by Aspergillus fumigatus commonly cause invasive pulmonary aspergillosis (IPA) in immuno-compromised patients. The development of the infection in immunodeficient patients demonstrates the importance of the host immune response in controlling aspergillosis. However, investigations of host-microbe interactions has been hampered by the lack of tools for their non-invasive assessment. We report on a multimodal approach to study the evolution of the infection (CT, BLI and MRI) and the response of the host’s immune system (19F MRI) simultaneously and longitudinally in vivo.

Methods

Four groups of Balb/c mice were studied: (1) non-infected mice receiving perfluorocarbon-particles (PFCE) [1]; (2-4) mice being infected with A. fumigatus (fLuc+, intranasal 106 spores), receiving PFCEs and either (2) no immune-suppression, (3) hydrocortisone acetate (HCA, 9mg/mouse) or (4) cyclophosphamide (CY, 200mg/mouse) [2]. Pulmonary infection was followed up by BLI (IVIS Spectrum, Perkin Elmer), CT (Skyscan 1278, Bruker) and MRI (ultrashort-echo MRI using a Bruker Biospec 94/20) according to [2, 3]. 19F MRI was performed with a purpose-built, double-tuned 1H/19F coil, commencing 4h post infection/ 1h post injection of Zonyl® FSP (Z-PFCE) particles(d0/d1) [4]. 19F and 1H MRI was performed daily. In vivo CT was performed on d1/d3. Ex vivo BLI was performed on d3 on the excised lungs.

Results/Discussion

Macrophages, identified by their characteristic high surface expression of CD11b and F4/80, phagocytosed ZPFCE-NPs without toxic effects.

In vivo data for the aspergillosis models (CY and HCA) and for non-infected mice are shown in Fig.1. A rapid influx of macrophages into the lung was seen in immunocompetent mice. Inflammation was quickly resolved with return to baseline levels after 24 hrs. In both models of immunosuppression, the immediate innate response to infection was reduced. In HCA-treated mice, the exacerbated intrusive recruitment of immune cells resulted not only in the labeling of tissue-resident macrophages but also dendritic cells, as indicated by elevated ZPFCE-NPs levels in cervical lymph nodes. Inflammation was less pronounced in the CY-treated mice.

Immune reaction was correlated with infection using BLI, CT and UTE MRI (Fig.2) No signs of infection were seen in immunocompetent mice. Mild lesion formation was seen in HCA- and strong lesion formation in CY-treated mice.

Conclusions

Our results demonstrate that CY-immunosuppression can lead to lethal infections (IPA) with a mild initial inflammation. HCA-based immunosuppression leads to less lesion formation but triggers acute inflammation leading to possibly lethal tissue destruction. For the first time, the dynamic profile of infection and inflammation was monitored in vivo and simultaneously in these aspergillosis models.

References

  1. Dewitte, H., Geers, B., Liang, S., Himmelreich, U., Demeester, J., De Smedt, S.C., Lentacker, I. (2013). Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and19F-MRI tracking of dendritic cells. J. Control. Release 169, 141–149.
  2. Poelmans, J., Hillen, A., Vanherp, L., Govaerts, K., Maertens, J., Dresselaers, T., Himmelreich, U., Lagrou, K., Vande Velde, G. (2016). Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging. Lab. Invest. 96, 692–704.
  3. Poelmans, J., Himmelreich, U., Vanherp, L., Zhai, L., Hillen, A., Holvoet, B., Belderbos, S., Brock, M., Maertens, J., Velde, G.V., Lagrou, K. (2018). A multimodal imaging approach enables in vivo assessment of antifungal treatment in a mouse model of invasive pulmonary aspergillosis. Antimicrob. Agents Chemother. 62, e00240–18.
  4. Saini, S., Korf, H., Liang, S., Verbeke, R., Manshian, B., Raemdonck, K., Lentacker, I., Gysemans, C., De Smedt, S.C., Himmelreich, U. (2019).
    Challenges for labeling and longitudinal tracking of adoptively transferred autoreactive T lymphocytes in an experimental type-1 diabetes model. MAGMA 32, 295–305.

Acknowledgement

We are grateful for the financial support by the following funding agencies: the European Commission Marie Curie (ITN) BetaTrain (289932), the European Horizon 2020 ‘PANA’ project under grant agreement n° 686009, the Research Foundation Flanders (FWO, G.0A75.14, G.0B28.14 and G.069115N), the Agentschap voor Innovatie door Wetenschap en Technologie for the SBO NanoCoMIT (IWT SBO 140061) and the European ERA-NET project ‘CryptoView’ (3rd call of the FP7 programme Infect-ERA).

Assessment of differential local immune response to infection by A. fumigatus by 19F-MRI

(A) 19F MRI and 1H MRI were obtained from hydrocortisone acetate (HCA), cyclophosphamide (CY) and infected-immunocompetent (I-IC) mice as well as non-infected control mice (N-IC) after systemic injection of ZPFCE-NPs on day 0 (4h) and day 1.

(B) Quantification of 19F MRI from the lung region by comparison with a reference (R in panel A) containing 30mM ZPFCE-NPs.

(C) 19F MRI signal was also observed from the lymph node region for the HCA group on day 1. Mean 19F MR signal intensity was quantified with respect to the 30mM reference placed next to each animal.

Assessment of lung infection.

(A) In vivo follow-up of A. fumigatus infected mice using computed tomography (CT) and 1H MR imaging. Mice were scanned on day 1 and day 3, post infection for the confirmation of pulmonary fungal infection progression using CT imaging where lesions in HCA (hydrocortisone acetate and CY (Cyclophosphamide) mice can be observed as compared to I-IC and non-infected immunocompetent (N-IC) group.

(B) Quantitative estimation of lung lesion volume based on UTE-MR images (mean±SEM).

Keywords: 19F MRI, Bioluminescence, CT, fungal infection, immune response