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.

<table><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>&lt;sup&gt;64&lt;/sup&gt;Cu-CuCl&lt;sub&gt;3&lt;/sub&gt;</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 &amp;amp; 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&quot; x 10&quot;</td></tr><tr><td>Autoradiography film</td><td>&amp;nbsp;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>&amp;nbsp;00H-2146-K0</td><td>5 &amp;mu;m SEC-s3000 400 &amp;Aring;, 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 ...

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Neuroscience

1.8K Views.  Stanford University. Deze techniek maakt gebruik van PET-beeldvorming om de in vivo distributie en dynamiek van B-cellen in het centrale zenuwstelsel op te helderen. Onze aanpak is gunstig voor het bestuderen van neurologische aandoeningen, waarbij het aangetaste ruggenmerg de analyse uitdagender maakt. Er zijn dus een paar belangrijke voordelen aan onze techniek.Ten eerste hebben we een nieuwe tool gemaakt om een pan-B-celbiomarker te volgen waarmee we een reeks B-celsubsets kunnen vastleggen en in vivo k...

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

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Immunology and Infection

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

Medicine

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

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

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

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

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

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

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Role of CD4 T Cell in Relapsing-Remitting Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic animal model for studying MS. In this study, we aimed to investigate the role of CD4 T cells in the initiation and relapse of EAE, focusing on the activation phase and immune response. To create the EAE mice model, female mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 emulsified with complete Freund&#39;s adjuvant (CFA). Clinical scores were assessed daily, and results demonstrated that mice in the EAE group exhibited a classic relapsing-remitting pattern. Hematoxylin-eosin (H&#38;E) and luxol fast blue (LFB) staining analysis revealed significant infiltration of inflammatory cells in the CNS and demyelination in EAE mice. Regarding the activation phase, both CD4+CD69+ effector T (Teff) cells and CD4+CD44+CD62L- effector memory T (Tem) cells may contribute to the initiation&#160;of EAE, however, the relapse stage was probably dominated by CD4+CD44+CD62L- Tem cells. Additionally, in terms of immune function, helper T (Th)1 cells are primarily involved in initiating the EAE. However, both Th1 and Th17 cells contribute to the relapse stage, and the immunosuppressive function of regulatory T (Treg) cells was inhibited during the EAE pathological process.

An experimental autoimmune encephalomyelitis (EAE) model in mice was employed to investigate the role of CD4 T cells in the initial and relapse of EAE from the perspective of the activation phase and immune effect function.

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Biology

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Tietz, S. M. et al. <a href="https://app.jove.com/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EAE ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Schmitz, K., et al. <a href="https://app.jove.com/v/55321">Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice.</a> J. Vis. Exp (2017).
In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that ultimately results in myelin damage, mimicking the demyelinating condition of multiple sclerosis.

1. EAE Induction in SJL/J Mice

	Mice
	
		Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = ...

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

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