Name: imaging cd19+ b cells in an experimental autoimmune encephalomyelitis mouse model using positron emission tomography
Uploaded: 2023-01-20T12:35:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography at JoVE.com

We are grateful for the support from the SCi3 small-animal imaging facility at Stanford and Dr. Frezghi Habte for his technical assistance with the PET/CT. LC-MS is performed by the core staff at the Stanford University Mass Spectrometry (SUMS) core facility and we appreciate the staff for providing this service. We thank Horizon Therapeutics for very kindly providing the hCD19-mAb and Jodi Karnell in particular for her technical guidance and support. This work was funded by the NIH NINDS (1 R01 NS114220-01A1).

<ol>
	<li>Dobson, R., Giovannoni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis+-+a+review.">Multiple sclerosis - a review.</a> European Journal of Neurology. 26 (1), 27-40 (2019).</li><li>Karussis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+of+multiple+sclerosis+and+the+various+related+demyelinating+syndromes:+A+critical+review.">The diagnosis of multiple sclerosis and the various related demyelinating syndromes: A critical review.</a> Journal of Autoimmunity. 48-49, 134-142 (2014).</li><li>Hauser, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+depletion+with+Rituximab+in+relapsing-remitting+multiple+sclerosis.">B-cell depletion with Rituximab in relapsing-remitting multiple sclerosis.</a> The New England Journal of Medicine. 358 (7), 676-688 (2008).</li><li>Chen, D., Gallagher, S., Monson, N. L., Herbst, R., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inebilizumab,+a+B+cell-depleting+anti-CD19+antibody+for+the+treatment+of+autoimmune+neurological+diseases:+Insights+from+preclinical+studies.">Inebilizumab, a B cell-depleting anti-CD19 antibody for the treatment of autoimmune neurological diseases: Insights from preclinical studies.</a> Journal of Clinical Medicine. 5 (12), 107 (2016).</li><li>Stevens, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+CD19+PET+tracer+for+detecting+B+cells+in+a+mouse+model+of+multiple+sclerosis.">Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis.</a> Journal of Neuroinflammation. 17 (1), 275 (2020).</li><li>Chaney, A. M., Johnson, E. M., Cropper, H. C., James, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+imaging+of+neuroinflammation+using+[11C]DPA-713+in+a+mouse+model+of+ischemic+stroke.">PET imaging of neuroinflammation using [11C]DPA-713 in a mouse model of ischemic stroke.</a> Journal of Visualized Experiments. (136), e57243 (2018).</li><li>Lyons, J. -. A., Ramsbottom, M. J., Cross, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+antigen-specific+antibody+in+experimental+autoimmune+encephalomyelitis+induced+by+recombinant+myelin+oligodendrocyte+glycoprotein.">Critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein.</a> European Journal of Immunology. 32 (7), 1905-1913 (2002).</li><li>Haugen, M., Frederiksen, J. L., Degn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cell+follicle-like+structures+in+multiple+sclerosis-With+focus+on+the+role+of+B+cell+activating+factor.">cell follicle-like structures in multiple sclerosis-With focus on the role of B cell activating factor.</a> Journal of Neuroimmunology. 273 (1-2), 1-7 (2014).</li><li>James, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+B+cells+in+a+mouse+model+of+multiple+sclerosis+using+64Cu-Rituximab+PET.">Imaging B cells in a mouse model of multiple sclerosis using 64Cu-Rituximab PET.</a> Journal of Nuclear Medicine. 58 (11), 1845-1851 (2017).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-mediated+methodology+for+the+site-specific+radiolabeling+of+antibodies+based+on+catalyst-free+click+chemistry.">An enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry.</a> Bioconjugate Chemistry. 24 (6), 1057-1067 (2013).</li><li>Lyons, J. -. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+are+critical+to+induction+of+experimental+allergic+encephalomyelitis+by+protein+but+not+by+a+short+encephalitogenic+peptide.">B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide.</a> European Journal of Immunology. 29 (11), 3432-3439 (1999).</li></ol>

The CD19 antibody was provided by Horizon Therapeutics.

This article describes a streamlined method for imaging human-CD19+ B cells in a mouse model of MS using CD19-PET. Due to the heterogeneous presentation of MS and varying responses to treatments, its management in the clinic can be challenging and new approaches for therapy selection and monitoring are greatly needed. PET imaging could serve as a powerful tool for monitoring disease progression and individual response to B cell-depleting therapy. In addition to MS, CD19-PET imaging could be used to monitor B cell depletion after treatment in subtypes of lymphomas and leukemia or other B cell mediated diseases. This protocol and representative data show the utility of imaging B cells in neurological diseases.
To study human CD19+ B cells in the context of MS, we chose the B cell-dependent MOG1-125 EAE model7. Similar to other EAE models, this model presents with symptoms of progressive paralysis and infiltration of immune cells into the CNS. However, the MOG1-125 model is unique in that it is a B cell-driven model: mice contain varying numbers of B cells in the subarachnoid space in the meninges, brain stem, parenchyma, and ventricles. These lymphocytes can be sparsely scattered throughout these regions and/or form follicle-like structures, which are also observed in humans with MS8,9. In addition to using na&#239;ve mice as controls, a complete Freund&#39;s adjuvant (CFA)-only induction kit may be used (i.e., an identical induction emulsion to what is given to EAE mice sans the MOG protein). In the EAE mouse model, the blood brain barrier (BBB) is dysfunctional and allows larger entities, such as antibodies, to cross. The CD19-mAb radiotracer will only bind and remain in the CNS if B cells are present; the tracer will circulate back into the blood pool if B cells are not present. We have demonstrated this using gamma counting and ex vivo autoradiography of CNS tissues by perfusing before measuring the radioactivity levels in the tissues. We have also demonstrated this in earlier publications reporting the use of mAb-based PET radiotracers (i.e., immunoPET imaging approaches) for detecting B cells in the CNS1,2.
The DOTA chelator was used since it has been used in clinical PET imaging with copper-64 labeled peptides and antibodies, and we aim to translate the hCD19-mAb for clinical imaging of MS patients. DOTA has adequate binding affinity to copper-64 in vivo. The in vivo stability is very important because free 64Cu goes to the liver and can obscure the signal of bound radiotracer; thus, it is important to measure the signal in the liver to calculate the relative signal compared to other organs. Muscle is typically taken as a control tissue, but in the case of EAE, there can be inflammation present in the muscles. The half-life of 64Cu is 12.7 h, which affords ample time for the DOTA-hCD19-mAb to bind to its target while ensuring signal can be measured by PET. When preparing the conjugate, small-scale (75-125 &#181;g) test reactions should be performed to determine the amount of DOTA to add to mAb to produce the desired DOTA/mAb ratio (e.g., a reaction of 6-10-fold excess DOTA-NHS-ester per mol mAb may afford a conjugate of 1-2 DOTA/mAb). The reaction time and temperature (e.g., 2-4 h or overnight at 4 &#176;C or room temperature) also influences the DOTA/mAb ratio and should be optimized. A titration with nonradioactive copper can be performed to calculate the number of DOTAs per mAb; however, we recommend performing MALDI-MS and/or LC-MS for more reliable and accurate results.
The calculated DOTA/mAb ratio is an average value for a particular sample and some variation is expected. For MALDI, several shots are taken per sample for the conjugated and unconjugated mAbs. We then calculate the ratio of conjugated to unconjugated to determine the average number of DOTA/mAb. The DOTA/mAb ratio is important because too many chelators will disrupt antibody binding and too few will lead to inconsistent radiolabeling and low signal. The ratio should be very close between batches of conjugate to maintain consistent signal intensity and binding kinetics; ideally, the same batch of conjugate should be used for all experiments within a particular study. A promising technique to reduce potential effects on immunoreactivity due to possible overconjugation is to use site-specific conjugation10 whereby the chelator conjugation is site-selective on the heavy chain glycans of the antibody, thus guaranteeing the addition of 1 chelator per mAb.
The radiolabeling reaction conditions should be optimized to ensure the highest labeling efficiency and yield since differences in antibody, DOTA/mAb ratio, and 64Cu molar activity, among other conditions, will impact radiolabeling. Using the optimal 64Cu to mAb conjugate ratio may allow the radiotracer to be used without purification, reducing the time required for radiolabeling and loss due to the gravity flow column and radioactive decay. A consistent and reliable molar activity may also be achieved when the same 64Cu to mAb conjugate ratio is used, which is especially important when comparing results across multiple cohorts of mice or imaging studies. The ITLC conditions may also be modified to suit each user. If purification is necessary, an aliquot should be saved for either HPLC and/or UV/Vis spectrophotometry so that the molar activity can be calculated.
It is important to note that using radiolabeled antibodies for imaging can be challenging. It is essential that the antibody used for the radiotracer be biologically inert so as not to have a physiological effect. Moreover, since antibodies have a long blood residence, one must wait long enough for circulation, binding, and clearance of a given mAb to ensure a suitable signal-to-background without compromising image quality. Typically waiting for 20-48 h for a 64Cu-labeled mAb is sufficient but one should image at 2, 4, 6, 12, 24, 48 h post injection when assessing a new mAb PET tracer to determine the best time point for imaging in a given rodent model. The same is true for acquiring ARG images with the highest signal-to-background ratio. The representative images in this protocol were taken at 18-20 h post injection, though other time points may be used depending on the radioisotope used. Different antibodies binding to different epitopes of CD19 will produce varying results and should be rigorously characterized.
When analyzing the spinal cord signal, it is important to position mice on their backs in the scanning bed to reduce movement caused by breathing. Additionally, supine placement can help straighten the spine in mice that have increased spinal curvature due to the progression of EAE disease. Another important aspect to consider when aiming to detect signal in the spine and spinal cord is to avoid injecting MOG1-125 on the flank as the injection sites can bind the tracer due to the associated immune response in those areas. The close proximity of the injection site can interfere with spinal cord analysis; thus, injections in the chest are preferable for the application described herein.
The image analysis techniques used are specific for CNS imaging. A brain atlas tool within the image analysis software affords reproducible and reliable results as long as the registration of PET and CT is accurate. Using the semi-automated 3D brain atlas and adjusting it to fit the skull of each mouse allows for consistent ROIs between animals. Since there is currently no automated or semi-automated approach for analyzing the signal in spinal cords, manual ROIs must be drawn. Notably, when quantifying CD19+ B cells (or any cell type present in both the bone marrow and spinal cord), it is critical to eliminate the signal arising from the spinal column and bone marrow as much as possible. The reason for this is that na&#239;ve mice are known to contain more CD19+ B cells in their bone marrow than EAE mice, in which B cells leave the periphery to infiltrate the CNS5,11. This bone marrow signal can obscure the true signal in the spinal cord.
To delineate true spinal cord signal while minimizing the contribution of signal from the spinal column and bone marrow, Otsu thresholding of the CT image can be used to make an immutable ROI for the spinal column. A separate spinal cord ROI can then be easily drawn within the spinal column. The same technique can also be applied to measure bone marrow in the femur. This is a very useful method to gain insights into tracer binding in the spinal cord. However, due to the relatively low spatial resolution of PET and issues pertaining to the partial volume effect when scanning small anatomical regions of mice, use of additional ex vivo confirmatory techniques (e.g., gamma counting, ARG) enables the validation of radiotracer binding in the spinal cord without the presence of blood, cerebrospinal fluid, or spillover signal from the spinal column.
Signal in the cervical/thoracic spinal cord tends to vary in the EAE mice depending on the severity of disease and how many B cells infiltrate during the adaptive immune response. This variation in the number of B cells that infiltrate, as well as the small amount of B cells in the CNS compared to those in the pelvic/spinal bone marrow of na&#239;ve mice, can make in vivo quantitation of spinal cord tissue challenging in mice. Given the spatial resolution of PET in small animal imaging, signal from the bone marrow can spill over onto the spinal cord signal. Ex vivo biodistribution and autoradiography completed here aid in validating the PET signal of the vertebrae versus spinal cord tissue. Mice are perfused prior to dissection to remove any unbound tracer in the blood pool so that the gamma counting and autoradiography results reflect the tracer that is actually bound in each organ rather than the tracer that is in the blood pool in that organ.
Radiotracers circulate through the blood, and with antibody tracers, specifically, there is often unbound radiotracer present in the blood for weeks after the initial injection. Since we are imaging the brain and spinal cord, which have many blood vessels, it is important to understand what portion of the signal is truly due to tracer binding in brain/tissue of interest versus that present in the blood pool. It is thus necessary to divide the brain signal by signal in the heart/blood pool. In the clinical setting, the same image analysis techniques of Otsu thresholding of vertebrae and ROIs of spinal cord tissues can be used for quantification. Given the larger tissue volumes in humans compared to mice, there should be significantly less impact from partial volume effects, leading to improved accuracy and negating the need for ex vivo techniques to confirm in vivo findings. The use of PET in the clinic will allow clinicians to personalize therapy for each patient depending on their individual B cell burden.
ARG is particularly useful for acquiring high-resolution images to enable more accurate delineation of the spatial location of tracer binding in small regions such as brain stem and cerebellum. The same sections and/or adjacent sections can be saved for immunohistochemical stains to confirm the presence of B cells. We have previously stained CNS tissues with CD45R/B220 (Supplemental Figure S1) to correlate the number of B cells with PET and ARG signal5,9. The staining can then be compared spatially to the ARG results to verify that the radiotracer signal matches the staining pattern. B cells can be present in clusters or diffusely throughout the brain stem; PET sensitivity is sufficiently high to measure the signal, which is encouraging for clinical translation. For spinal cord ARG, removing the spinal cord from the vertebrae ensures that the signal measured is due to tracer binding in the spinal cord tissue rather than the bone marrow and/or blood, which can obscure PET images due to partial volume effects.
Similar to ARG, ex vivo gamma counting enables the quantification of radioactive signal in individual organs. For this particular technique, it is important to measure the wet weight of tissues and ensure they are at the bottom of their respective tubes before placing the tubes in the gamma counter. The tubes must be labeled with the mouse number and tissue, so that the correct tube is used; the tube is then weighed on a calibrated balance and organs are inserted to the nearest tenth of a microgram (0.0001 mg). Some tissues are extremely small and the difference in the tube mass before and after will be in the order of 0.0001 mg. The tissues should be weighed immediately following dissection to prevent loss of moisture, which results in a lower mass. After weighing, the brain and spinal cord tubes should be filled with PBS to prevent from drying before freezing these tissues for ARG.

MS is an immune-mediated neurological disorder; the unique presentation in each patient can make management challenging for both patients and clinicians1. The disease itself is characterized by the presence of demyelinating lesions and immune cell infiltration in the brain and spinal cord, resulting in physical and cognitive impairment2. The traditional paradigm that MS is a T cell-mediated disease was first challenged in a landmark phase II clinical trial of rituximab3, a therapy targeting the CD20+ subset of B cells. Additional B cell therapies have since been developed that target CD194, a pan B cell biomarker that is expressed on a broader range of B cells, which can be both diagnostically and therapeutically advantageous. Moreover, existing methods to assess treatment efficacy (i.e., monitoring the number of relapses and magnetic resonance imaging [MRI] activity) do not afford early measures of response-thus placing patients at significant risk of CNS damage due to suboptimal therapy selection and optimization. Hence, there is a critical need for strategies to monitor specific immune cells, such as CD19+ B cells, in real time in the CNS and periphery of MS patients.
PET imaging is a robust imaging technique that allows in vivo, whole-body visualization of a given target of interest, such as CD19. While blood draws, records of relapse rates, and lesion monitoring via MRI provide snapshots into treatment efficacy, PET imaging can allow researchers and clinicians to monitor effectiveness of a therapy across the entire body. This proactive approach to therapeutic monitoring allows clinicians to assess medication effectiveness in real time, enabling rapid adjustments as needed. Monitoring the location and density of cell populations associated with disease also permits longitudinal assessment of severity using patient-specific anatomic information. It is thus essential to establish reproducible analytical methods to reliably utilize the full potential of PET imaging in clinical and preclinical settings.
This paper describes methods (Figure 1) to perform PET imaging, ex vivo gamma counting, and autoradiography (ARG) of CD19+ B cells with a 64Cu-labeled anti-human CD19 monoclonal antibody (mAb), known as 16C4-TM (64Cu-hCD19-mAb), in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS induced in transgenic mice expressing human CD19 (hCD19) using human recombinant myelin oligodendrocyte glycoprotein 1-125 (MOG1-125). We also provide methods to assess radiotracer binding accurately and reproducibly in the brain and spinal cord, both critical sites of pathogenesis often severely affected in this and other neurodegenerative models. These techniques allow for non-invasive investigation of the role of B cells in disease pathology and have the potential to be clinically translated to assess the efficacy of anti-B cell therapies in MS.

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	<tbody>
		<tr>
			<td>0.5 mL 50 kDa MWCO Centrifugal filter</td>
			<td>MiliporeSigma</td>
			<td>UFC505008</td>
			<td>centrifugal filter</td>
		</tr>
		<tr>
			<td>64Cu-CuCl3</td>
			<td>Washington University in St. Louis; University of Wisonsin, Madison; or another vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AR-2000 Radio-TLC Imaging Scanner</td>
			<td>Eckert &#38; Ziegler</td>
			<td>AR-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoradiography cassette</td>
			<td>Cole Palmer</td>
			<td>EW-21700-34</td>
			<td>Aluminum, 8&#34; x 10&#34;</td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>&#160;GE Life Sciences</td>
			<td>28-9564-78</td>
			<td>Storage Phosphor Screen BAS-IP SR 2025 E Super Resolution, 20 x 25 cm, screen only</td>
		</tr>
		<tr>
			<td>Butterfly Needle Catheter</td>
			<td>SAI Infusion Technologies</td>
			<td>BLF-24</td>
			<td></td>
		</tr>
		<tr>
			<td>DOTA-NHS-ester</td>
			<td>Macrocyclics</td>
			<td>B-280</td>
			<td></td>
		</tr>
		<tr>
			<td>EAE Induction Kit</td>
			<td>Hooke Laboratories</td>
			<td>EK-2160</td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger Counter</td>
			<td>Ludlum</td>
			<td>14C</td>
			<td></td>
		</tr>
		<tr>
			<td>GNEXT PET/CT Scanner</td>
			<td>Sofie</td>
			<td>GNEXT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hidex Automatic Gamma Counter</td>
			<td>Hidex</td>
			<td>AMG</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Column</td>
			<td>Phenomenex</td>
			<td>&#160;00H-2146-K0</td>
			<td>5 &#956;m SEC-s3000 400 &#197;, 300 x 7.8 mm</td>
		</tr>
		<tr>
			<td>Illustra NAP-5 column</td>
			<td>Cytiva</td>
			<td>17085301</td>
			<td>DNA gravity column</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td></td>
			<td>ARG analysis software</td>
		</tr>
		<tr>
			<td>Low Protein Binding Collection Tubes (1.5 mL)</td>
			<td>Thermo Scientific</td>
			<td>PI90410</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Lite Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840281400</td>
			<td>UV-Vis micro/nano-spectrophotometer</td>
		</tr>
		<tr>
			<td>PCR tubes 0.2 mL, for DNA grade</td>
			<td>Eppendorf</td>
			<td>30124707</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon phosphor imager 9410</td>
			<td>GE Healthcare</td>
			<td>8149-30-9410</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoQuant</td>
			<td>Invicro</td>
			<td>Version 4 Patch 3</td>
			<td>PET Analysis Software; must purchase brain atlas add-on</td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>PI89882</td>
			<td>Desalting column</td>
		</tr>
	</tbody>
</table>

imaging cd19+ b cells in an experimental autoimmune encephalomyelitis mouse model using positron emission tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

The hCD19-mAb was DOTA-conjugated and radiolabeled with 64Cu as shown in Figure 2. EAE and na&#239;ve mice underwent PET/CT scanning (Figure 3) 18-24 h after injection with 64Cu-DOTA-hCD19-mAb. PET/CT images were co-registered using the PET analysis software, and the CNS tissues were analyzed using manual ROIs or a semi-automated 3D brain atlas. Radiotracer binding in ROIs (Figure 4) was higher in EAE mice than in na&#239;ve mice. Ex vivo gamma counting and ARG showed increased binding in the spinal cord (both lumbar and cervical thoracic segments) and brain (ARG only) of EAE mice compared to na&#239;ve (Figure 5 and Figure 6). Ex vivo gamma counting of perfused mice also showed decreased radiotracer binding in peripheral organs, including spleen, femur, and bone marrow (Figure 5), consistent with B cells leaving the periphery and infiltrating the CNS in this EAE model.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64133/64133fig02.jpg" /> 
Figure 2: Conjugation and radiolabeling scheme for generating 64Cu-labeled human-specific CD19 monoclonal antibody, 16C4-TM mAb (64Cu-DOTA-hCD19-mAb), in addition to quality control data. (A) Reaction of DOTA-NHS-ester with hCD19 monoclonal antibody to produce hCD19-DOTA conjugate (not to scale) and radiolabeling with 64Cu-CuCl3 to produce 64Cu-DOTA-hCD19-mAb.&#160;(B) Representative ITLC chromatograph. The peak at 40-60 cm is the radiolabeled antibody; unbound 64Cu-CuCl3 travels with the mobile phase and would be present from 200 to 240 cm. There is no detectable free 64Cu-CuCl3 in this chromatograph. (C) The quality control specifications of the radiolabeled antibody. Abbreviations: DOTA-NHS ester = 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester; ITLC/HPLC = instant thin layer chromatography/high-performance liquid chromatography; MALDI/LC-MS = matrix-assisted laser desorption/ionization/liquid chromatography-mass spectrometry; CPM = counts per minute. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64133/64133fig03.jpg" /> 
Figure 3: Photographs demonstrating how to secure mice in a 3D-printed bed within the PET scanner to enable high quality imaging of the spinal cord and brain while minimizing motion. (A) 3D printed four-mouse scanner bed (also known as &#34;mouse hotel&#34;) equipped with heating elements and anesthesia tubing. (B) Anesthetized mice in a supine position to maximize straightness of the spine; the bed position of each mouse is recorded. (C) Mice taped securely across their head to minimize motion in the brain and across the belly to minimize motion from breathing, without affecting breathing. (D) Mouse bed positioned within the scanner and taped to the scanning bed. Anesthesia tubing was connected from scanner to bed and isoflurane set to 2%. Mouse breathing was monitored to ensure appropriate isoflurane level before closing the scanner door. Abbreviation: PET = Positron emission tomography. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64133/64133fig04.jpg" /> 
Figure 4: Spinal cord image and brain analysis and results using PET analysis software. (A) i) ROIs (pink and tan) drawn on spine to separate lumbar from the thoracic and cervical vertebrae and prepare image for Otsu thresholding. ii) Spinal vertebrae (turquoise and red) were segmented out using Otsu Thresholding. iii) Vertebrae were then made immutable in the 3D ROI menu, and the spinal cord divided into cervical/thoracic (purple) and lumbar (navy) ROIs. iv) The vertebral ROI was removed, leaving spinal cord ROIs and representative brain atlas applied. (B) Representative analysis of PET results from various CNS regions represented as %ID/g normalized to the ROI of the heart within each animal. PET acquisition was a 10 min static scan via PET/CT imaging. Brain regions quantified using a semi-automated brain atlas approach, shown in panel A. iv) Representative results show either significance or trending toward significant increase in tracer binding in the brain and thoracic spinal cord. Statistics performed using Student&#39;s t-test (*: p &#60; 0.0332). Abbreviations: PET = Positron emission tomography; ROIs = regions of interest; CNS = central nervous system; CT = computed tomography; %ID/g = percent injected dose per gram of tissue; EAE = experimental autoimmune encephalomyelitis. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64133/64133fig05.jpg" /> 
Figure 5: Representative quantification of ex vivo gamma counting in various organs in EAE and na&#239;ve mice expressed as %ID/g. Post-PET scan, mice were perfused with PBS to remove the radiotracer present in the blood, either free or bound to blood-resident CD19+ B cells, and organs quickly dissected and weighed to have an accurate weight of each organ. Tracer binding is significantly decreased in the spleen and bone marrow in EAE mice compared to na&#239;ve ones. Increased radiotracer binding is observed in both the lumbar and cervical/thoracic spinal cord segments of EAE mice. The brain does not show significant increase in radiotracer signal, though it is trending toward significant increase. Statistics performed using Student&#39;s t-test (*: p &#60; 0.0332; ****: p &#60; 0.0001). Abbreviations: PET = Positron emission tomography; %ID/g = percent injected dose per gram of tissue; EAE = experimental autoimmune encephalomyelitis; PBS = phosphate-buffered saline. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64133/64133fig06.jpg" /> 
Figure 6: Ex vivo ARG images depict 64Cu-DOTA-hCD19-mAb binding in sagittal brain sections and whole spinal cords from EAE compared with naive mice. Digital phosphor storage films were scanned using a phosphor imager after being exposed to radioactive tissue samples for approximately 10 half-lives (127 h or 5 days). The resulting images reveal visually higher signal in the brain of EAE mice compared to brain sections from na&#239;ve mice, which is expected due to the regions known to contain B cells in this model5. Specifically, there is increased tracer signal in the brain stem, cerebellum, and ventricles of EAE mouse brain sections. This increase in signal for EAE mouse brain sections mirrors what was found for in the whole-brain PET quantification detailed above. Similarly, there is an increase in radiotracer binding in both the cervical/thoracic and lumbar spinal cord segments compared to na&#239;ve spinal cords, reflecting what was found using ex vivo gamma counting. Abbreviations: PET = Positron emission tomography; EAE = experimental autoimmune encephalomyelitis; Vent = ventricles; Cb = cerebellum; BS = brain stem; TSc = thoracic and cervical spinal cords combined; LSc = lumbar spinal cord. <a href="https://www.jove.com/files/ftp_upload/64133/64133fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Staining of CNS tissues of na&#239;ve and EAE mouse brain tissue with CD45R/B220. B cells are observed in brainstem, meninges, and white matter of EAE mice (n = 7 EAE, n = 5 na&#239;ve mice, average four slices per animal). This figure is from 5. Scale bars = 5 mm (low magnification [1x]) in sagittal brain images, 100 &#181;m (high magnification [20x]) in brainstem, meninges, and cerebellar white matter. <a href="https://www.jove.com/files/ftp_upload/64133/Suppl Fig 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography at JoVE.com

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

This technique uses PET imaging to elucidate the in vivo distribution and dynamics of B cells in the central nervous system. Our approach is beneficial for studying neurological diseases, where the affected spinal cord makes analysis more challenging. So there are a couple of main advantages to our technique.First, we have created a new tool to track a pan B cell biomarker that allows us to capture a range of B cell subsets and visualize them in vivo. Secondly, our spinal cord analysis method enables highly accurate and reproducible quantification of PET signals in this region. The great thing about our technique is that it is disease-agnostic.It can be used from preclinical to clinical PET imaging in any scenario, where B cells, and/or the spinal cord are of interest. 18 to 24 hours after radiolabeling and injecting the antibody into the mouse, prepare the mouse for scanning by applying eye gel to the eyes. Ensure the four-mouse scanning bed is equipped with a heating pad with isoflurane set from 1.5 to 2%Place the mouse in a supine position on the scanning bed, and gently pull the mouse tail to straighten the spine.Once the mouse is in a supine position, securely tape it with soft microscope tape over the head and belly to minimize the motion from breathing. Make a record of the scanning position for each mouse in a lab notebook. After securing the first group, close the bed, and check the bed placement by running a CT scan.Click on CT Center Field of View, and once the bed is in position, run the CT test scan to ensure the placement is correct. Repeat until the bed position is satisfactory. Place a small white tape on the scanner bed to mark the correct bed placement for the remainder of the study.Open the Motion Controller menu, and click on PET Center Field of View to move the mice into the PET ring. Once the bed is in the PET ring, initiate the scanning sequence by clicking on Run, and wait for the scanner to automatically complete the PET scan, and move from the PET ring to the CT for CT acquisition. Since experimental autoimmune encephalomyelitis, or EAE, mice have pronounced curvature of the spine due to disease progression, scanning them while on their backs helps to straighten the spine.Make an incision down the dorsal side of the animal, and remove the skin and fur to expose the spinal column. Cut along three transverse planes through the spinal column at the neck, directly under the rib cage, and at the top of the pelvis to separate the lumbar from the cervical and thoracic regions. Carefully remove the segmented spinal column to get two pieces, lumbar and cervical thoracic.Then isolate the lumbar spinal column by carefully trimming the spinal column from the pelvic end until the lumbar spinal cord is visible. To expel the lumbar spinal cord, use a slip-tip syringe filled with PBS and create a seal between the syringe and the spinal column using the thumb and forefinger. Gently push the PBS through the syringe to expel the spinal cord onto an absorbent pad, and repeat for cervical thoracic spinal cord by inserting the syringe from the cervical side.Place the spinal cord tissues in a gamma counting tube. Record the dry weight, and add PBS to ensure the tissue is at the bottom of the tube to avoid drying. Place the tube on ice until ready for counting.To begin the analysis of regions of interest in the spinal cord, open the 3D Region of Interest Tool from the Navigation menu. Under the Regions of Interest header, use the plus sign at the bottom of the menu to create six regions of interest, lumbar region of interest, cervical thoracic region of interest, lumbar skeleton, thoracic skeleton, lumbar spinal cord, thoracic spinal cord. To avoid visual interference from the PET signal, click on F3 to turn off the PET.Go to the top of the 3D Region of Interest Tool Operator, and click on the solid dot at the right of the cursor symbol to open the 3D Paint Mode, and Erode/Dilate menu. Select Sphere, and change the size to 20 pixels. Likewise, set dilate to plus five.Before proceeding further, go to the bottom of the menu, and ensure the lumbar region of interest is selected. On the CT, find the L6 vertebra of the spinal column. Starting with one vertebra above L6, draw a rough lumbar region of interest over the five vertebrae above the hips.Then switch to cervical thoracic region of interest, and trace the remainder of the spine to the base of the skull. After drawing the generalized regions of interest, go to the top of the operator, and select the Segmentation Algorithms menu. From the dropdown menu, select Otsu Thresholding, then select lumbar region of interest for the input, and ensure lumbar skeleton is chosen at the bottom of the menu.In the dropdown menu next to Image, ensure the CT scan is selected, indicated here by the number zero. Click on Apply, and repeat the procedure for cervical thoracic region of interest and thoracic skeleton. After using Otsu Thresholding to create the skeleton regions of interest, return to the Navigation menu, and delete the region of interest, or check mark the H column for both the rough lumbar and cervical thoracic regions of interest to hide them.Check mark the I column for both skeletal regions of interest, so they cannot be edited. Finally, return to the top of the 3D Region of Interest Tool Operator, and go to the 3D Paint menu to draw the spinal cord regions of interest. Select the Sphere tool again, and trace the spinal cord within the skeleton for both the lumbar and thoracic, ensuring the correct region of interest is selected at the bottom of the menu.To erase any region of interest, click on Command/Control and draw over the part to be erased. Check the spinal cord region of interest from all three planes to ensure no region of interest is drawn outside the spinal column. If the PET signal was turned off, press F3 after the spinal cord regions of interest are drawn to turn the PET back on, or select the Visual Controller, and click on the PET bar.Go back to the Navigation menu. Click on the Grid icon to show table. Copy the table into spreadsheet software, and Save the file.PET imaging revealed elevated radiotracer binding in the brain and thoracic spinal cord of EAE mice compared to the naive mice. Ex vivo gamma counting showed increased binding in both lumbar and cervical thoracic spinal segments, and the brain of EAE mice compared to naive. Ex vivo autoradiography images showed increased radiotracer binding in sagittal brain sections, specifically in the brainstem, cerebellum, and ventricles of EAE mice compared to naive mice.Similarly, an increased radiotracer binding was observed in both the cervical thoracic and lumbar spinal cord segments of EAE mice compared to naive spinal cords. After PET imaging and gamma counting, we can investigate the relationship between PET signal and the target of interest through molecular biology techniques, such as flow cytometry and immunohistochemistry. Our technique has paved the way for researchers to ask questions about the in vivo role of B cells in multiple disease areas, including stroke, multiple sclerosis, other autoimmune diseases, and cancer.

Research

JoVE Journal

Neuroscience

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Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

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Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

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Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

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Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

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