(2024) 21:161
Smith et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03127-2

Journal of Neuroinflammation

Open Access

RESEARCH

Astrocytic stress response is induced
by exposure to astrocyte‑binding antibodies
expressed by plasmablasts from pediatricpatients with acute transverse myelitis
Chad Smith1, Kiel M. Telesford1, Sara G. M. Piccirillo2,3, Yamhilette Licon‑Munoz2,3, Wei Zhang1, Key M. Tse1,
Jacqueline R. Rivas1, Chaitanya Joshi1, Dilan S. Shah1, Angela X. Wu1, Ritu Trivedi1, Scott Christley4, Yu Qian5,
Lindsay G. Cowell4, Richard H. Scheuermann5,6, Ann M. Stowe7, Linda Nguyen1, Benjamin M. Greenberg1 andNancy L. Monson1,8*Abstract
Background Pediatric acute transverse myelitis (ATM) accounts for 20–30% of children presenting with a firstacquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple
sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B
cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest
B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies
that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection,
and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease.
Methods We compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy
controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk
IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated
from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI)
in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential
impact of these rhAbs on astrocyte health by measuring stress and apoptotic response.
Results We found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autore‑
activity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autore‑
activity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocytebinding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric
ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual
antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to theseastrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis.
Conclusions Discordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.

*Correspondence:
Nancy L. Monson
Nancy.Monson@UTSouthwestern.edu
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Smith et al. Journal of Neuroinflammation(2024) 21:161

Background
Pediatric acute transverse myelitis (ATM) is an immunemediated inflammatory disease of the spinal cord with anincidence rate of 1.7–2 pediatric cases per million children per year [7, 13, 46]. ATM typically presents withlimb weakness, sensory deficits, and/or bladder/bowel
dysfunction evolving over hours to days. Magnetic resonance imaging (MRI) is the primary tool for diagnosis of
ATM, particularly as cerebrospinal fluid (CSF) protein
and cell counts can be normal in 20–50% of pediatric
ATM cases [2]. ATM accounts for 20–30% of children
presenting with a first acquired demyelinating syndrome,and can occur as an isolated syndrome, known as idiopathic TM. However, it can also be the first clinicalpresentation of other acquired demyelinating syndromes(ADS), including multiple sclerosis (MS), aquaporin4-IgG positive neuromyelitis optica spectrum disorder(NMOSD) or myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) diagnosis [3, 7].
Compared to adults, children with ATM appear to havea lower incidence of MS. Epidemiological studies haveshown approximately one-third of adult patients presenting with ATM evolve to a diagnosis of MS [15, 22, 28,
35, 39] while only 22% of pediatric patients evolve to adiagnosis of MS [8, 18]. Additionally, compared to adults,
children usually improve within 2 weeks following acuteimmunotherapy (eg corticosteroids, IVIG and plasmaexchange) and have better outcomes, with nearly one-halfmaking a complete recovery by 2 years [2]. One possible
difference could be variations in the composition of lymphocytes involved in the autoimmune pathology of children compared to adults.
B cells in particular, have been implicated in the pathogenesis of CNS autoimmune diseases, including MS. CSF
or blood B cell biomarkers to identify the extent of neuronal and glia inflammation, stress and irreversible injury
are however lacking in ATM. We previously demonstrated that plasmablasts (PBs), the earliest B cell subtype
producing antibody, are expanded in adults with ATM[30]. Plasmablasts are a recently activated antigen-experienced B cell subtype whose default program is to produce
high affinity neutralizing antibodies against non-self antigens [4], mature into long-lived plasma cells, and hometo the bone marrow where they can produce antibodies
for decades [38]. Autoreactive PBs are typically undetectable in healthy individuals, but in the context of adultATM, 28% of PBs that we isolated from the expanded PB
pool produce self-reactive antibodies that target cytoplasmic antigens within neurons. These autoreactive PBs
also tend to utilize antibody variable heavy chain 4 (VH4)family genes.
Little is known about B cell involvement in pediatricATM, although altered B cell subset distributions are

Page 2 of 16

reported in pediatric MS [44]. Therefore, we investigatedpediatric ATM patients and healthy controls to profile B
cells for evidence of involvement in CNS autoimmunity
associated with pediatric ATM. We found that the frequency of peripheral blood PBs was reduced in pediatric
ATM patients compared to pediatric HC. Pediatric ATM
patients had a similar frequency of VH4 + PB comparedto pediatric HC. Most autoreactive PBs from pediatric
ATM patients recognized astrocytes in CNS tissue andprimary human astrocytes in cell culture. Accumulationof somatic hypermutation into the antibody heavy chain
genes increased binding intensity to astrocytes. Exposure of human primary astrocytes to astrocyte-binding
antibodies cloned from PBs of pediatric ATM subjects
induced astrocytic stress, but did not advance to apoptotic events.

Materials and methods
Human blood acquisition and processing

All subjects and/or their legally authorized study partners signed the written informed consent approved by
the Institutional Review Board of the UT Southwestern
Medical Center (UTSW), in accordance with the Federalwide Assurance on file with the Department of Healthand Human Services (USA). The age, gender, diagnosis,
and other patient information is reported in Table 1.
Treatment-naïve pediatric ATM patients at high risk fordeveloping MS (ages 3–13) and pediatric healthy controls (HC) (ages 3–12) were included and were matchedby age. As pediatric ATM cases are rare, no power analysis was performed to determine the number of subjects needed to detect a difference between the groups.
Table 1 Pediatric subject information
Patient code Age Gender Current
Diagnosis

Oligoclonal Ig index
band status

PTM-A

12

F

ATM

0

Unknown

PTM-B

4

F

ATM

0

0.63

PTM-C

13

F

ATM

0

0.59

PTM-D

10

F

ATM

0

0.47

PTM-E

13

F

ATM

Unknown

Unknown

PHC-A

12

F

Healthy

NA

NA

PHC-B

6

F

Healthy

NA

NA

PHC-C

9

F

Healthy

NA

NA

PHC-D

5

F

Healthy

NA

NA

PHC-E

4

F

Healthy

NA

NA

PHC-F

11

M

Healthy

NA

NA

PHC-G

10

M

Healthy

NA

NA

PHC-H

10

M

Healthy

NA

NA

PHC-I

7

F

Healthy

NA

NA

PHC-J

3

F

HealthyNA

NA

Smith et al. Journal of Neuroinflammation(2024) 21:161

Page 3 of 16

Pediatric patients were classified as high risk for MS ifthey presented with at least one non-enhancing whitematter lesion by MRI and/or a high Ig index in the cerebrospinal fluid. None of these patients had evidenceof autoantibody titers to MOG or AQP4. We collected
whole blood in Acid Citric Dextrose (ACD) tubes for thisstudy. After collection, ACD tubes were centrifuged tocollect plasma and the non-plasma portion subjected to
Ficoll centrifugation to isolate peripheral blood mononuclear cells (PBMCs). Plasma was stored in aliquots
at -80˚C and PBMCs were stored in aliquots at -140˚C.
Immunogenetics of pediatric TM and HC were compared to adult CIS patients at high risk for developing MS
presenting with transverse myelitis, which were reported
previously [39].

collected on days 3, 5, 7 and 10. The cell pellets werespun down and supernatants were passed through0.2um filters and subjected to antibody purificationon the NGC QUEST 10 system. The concentration of
the antibodies were determined by sandwich ELISA as
done previously [29, 37–39].

Mass cytometry (CyTOF)

Fluorescent immunohistochemistry

Cryopreseved PBMCs were thawed in complete RPMI,
counted, filtered, and treated with cisplatin (2.5μM,
Enzo Life Sciences, Farmingdale, NY, USA) in serum free
RPMI to confirm viability [14]. Cell staining for mass
cytometry was performed as described previously [9],using antibody panels in table S6. Samples were masschannel barcoded, pooled, and stained as a single sample [56]. Readouts were recorded on a CyTOF2 mass
cytometer (Fluidigm, South San Francisco, CA, USA).
Data were analyzed in Cytobank (cytobank.org) and with
FlowJo software (flowjo.com), including viSNE [6], and
FlowSOM [49] analysis.

At the height of EAE symptoms, mice were perfusedtranscardially with PBS and 4% PFA, and their brains
and spinal cords were extracted. After a further 48 h
post-fix with 4% PFA, tissue was washed with PBS
and embedded in paraffin. Sagittal brain slices andtransverse cord slices (5 μm) were obtained by the UTSouthwestern Histo Pathology Core. Slices were deparaffinated with 2 × 10 min washes with xylenes (Fisher),then washed with successive ethanol solutions: 2 × 100%
ethanol, 95% ethanol, 70% ethanol, 50% ethanol, 30%
ethanol. Slices were washed with PBS for 3 min, then
1% Glycine (Sigma) for 15 min. Slices underwent antigen retrieval by submersion in boiling Citrate Retrieval
Buffer (Vector BioLabs H-3300) in a pressure cooker
for 5 min. Slides were washed 2 × 5 min with PBS, thenwith 0.2% Triton X-100 (Sigma) for 10 min. Slides were
blocked for 2 h with 5% Goat Serum (ThermoFisher
50062Z) + 0.1% Triton X-100 in a humidified chamber,then incubated overnight at 4C in a humidified chamber with primary antibodies diluted in blocking buffer:
rhAbs (20 μg/mL), purified human IgG (2 μg/mL), Rabbit anti-GFAP (1:100 Abcam ab16997), Mouse antiMAP2 (1:200 ThermoFisher MA5-12826). The next day,slides were washed 3 × 5 min with 0.025% Triton X-100,
then incubated for 1 h at RT in a humidified chamberwith secondary antibodies diluted in blocking buffer:
Goat anti-Human conj AlexaFluor 488 (1:1000 ThermoFisher A-11013), Goat anti-Rabbit conj AlexaFluor
568 (1:1000 Abcam ab175471), Goat anti-Mouse conj
AlexaFluor 647 (1:500 Abcam ab150115). The rinses
were repeated, then slides were counterstained with
DAPI. Slides were washed twice with PBS and coverslips were mounted with Fluoromount G (ThermoFisher 00-4958-02). Slides were visualized on a Zeiss
LSM780 confocal microscope with a 40 ×/1.45NA oil
immersion lens.

Single‑cell antibody immunogenetics

Individually sorted antigen-experienced CD19 + CD27
+ CD38+ B cells from the blood were lysed, mRNA
was reverse-transcribed, and immunoglobulin variable
regions were amplified with multiple rounds of PCR as
previously described (32). Sanger sequencing was performed at the UTSWMC (University of Texas Southwestern Medical Center) sequencing core to generate
the antibody variable domain reads. Sequences wereanalyzed using the VDJServer analysis portal (33). V, D,
J gene calls, FR and CDR regions were annotated using
IgBlast v1.14.1 (34) with VDJServer IMGT 2019.01.23
germline database.
Recombinant human antibody cloning, expression,and isolation

HEK293T cells (ATCC, Manassas, VA) were maintained in HyClone Dulbeccos Modified Eagles Medium(DMEM) (GE Healthcare Life Sciences). All recombinant human antibodies (rhAbs) were transiently transfected into HEK293T cells with the lipid transfection
reagent JetPEI (PolyPlus Transfection) as done previously [29, 39]. Supernatants from these cultures were

Purification of IgG from plasma

To purify bulk IgG from plasma samples obtained from
ACD whole blood collections, we employed protein G
chromatography (Cytiva Life Sciences). We quantified
pure IgG preparations with an in-house IgG ELISA to
facilitate normalization across all samples for assays.

Smith et al. Journal of Neuroinflammation(2024) 21:161

Human astrocytes

Human astrocytes were purchased from ScienCell
Research Laboratories (Carlsbad, CA), grown in DMEM
(Sigma-Aldrich, St. Louis, MO, USA) and supplemented
with 10% heat-inactivated fetal bovine serum (Gemini
Bio-Products, Sacramento, CA, USA), 1% 100 × Penicillin–Streptomycin (Sigma-Aldrich, St. Louis, MO, USA)and 1% 200 mM l-glutamine (Sigma-Aldrich, St. Louis,
MO, USA).
Fluorescent immunocytochemistry

Cells were plated on laminin-coated coverslips (Sigma
L2020) and rested overnight in an 37 °C/5% ­CO2 incubator. The next day, cells were fixed with ice-cold 4% PFA for
10 min, then washed with PBS for 5 min. Cells were washedwith 0.2% Triton X-100 + 2 mg/mL BSA (Sigma A9647) for
10 min, then blocked with 0.1% BSA + 1% Goat Serum + 3%
BSA for 2 h. Cells were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer: rhAbs (20 μg/mL), Rabbit anti-GFAP (1:200). The next day, cells were
washed 4 × 3 min with 0.05% Triton X-100 + 1% Goat
Serum + 1% BSA, then incubated for 1 h at RT with secondary antibodies diluted in blocking buffer: Goat anti-Humanconj AlexaFluor 488, Goat anti-Rabbit conj AlexaFluor 568.
The washes were repeated, then cells were counterstainedwith DAPI and washed twice with PBS. Coverslips were
mounted on glass slides with Fluoromount G, then visualized on a Zeiss Axioscope.A1 fluorescent microscope with a
20×/0.45NA lens.
Commercial cell based assays

For measuring reactivity of rhAbs to AQP-4, we usedcell-based assays according to the manufacturer’s
instructions (Euroimmun FA1128-1010-50). This assay
expresses AQP-4 in fixed HEK293 cells [52]. For measuring reactivity to Hep-2 cells, we used a cell-based assay
according to the manufacturer’s instructions (Aesku.Bion
ANK-120). This assay consists of fixed Hep-2 cells. In
both assays, the rhAbs were diluted to 20 μg/mL in 0.1%
Tween-20 (Fisher) + 1% Goat Serum and cells were counterstained with DAPI. Cells were imaged as describedabove and quantified with FIJI. Positive staining of a rhAb
was defined as fluorescence intensity equal to or greaterthan the mean fluorescent intensity of the negative control plus 20 standard deviations.
Internalization procedure

After plating cells and resting overnight, rhAbs werediluted in 6 mM Glucose (Sigma) + 1% BSA + 2% Fetal
Bovine Serum + 1 mM Pyruvate (Gibco 111360-070) andadded to cell cultures at 20 μg/mL. After incubation, cells
were washed 3 × with ice-cold PBS, then fixed with 2%
PFA + 0.5% Triton X-100 for 2 min. Of note, exposure ofPage 4 of 16

the cells to Triton X-100 does not occur until after theinternalization step is complete. Cells were fixed for an
additional 15 min with 4% PBS, then washed 3 × with
PBS. Cells were blocked with 0.01% saponin (Sigma
47036) + 2% BSA + 0.1% lysine (Sigma 62840) for 30 min,then incubated with primary antibodies in blocking
buffer for 1 h: Rabbit anti-Clathrin (1:100 Cell Signaling
4796S), Mouse anti-EEA1 (1:100 Cell Signaling 48453S).
Cells were washed 6 × 5 min with blocking buffer, thenincubated with secondary antibodies and DAPI in blocking buffer for 1 h: Goat anti-Human conj AlexaFluor 488,
Goat anti-Rabbit conj AlexaFluor 568, Goat anti-Mouse
conj AlexaFluor 647. Cells were washed 6 × 5 min with
PBS, then fixed for 10 min with 4% PFA and washed 2
more times. Coverslips were mounted with FluoroMount
G and visualized with a Nikon CSU-W1 spinning disk
confocal microscope.
Quantification of autoreactivity by rhAbs

Autoreactivity was quantified using Fiji [43]. Cells wereidentified by using the Threshold function to detect
nuclei and were added to the Region of Interest (ROI)
manager. The mean fluorescence intensity (MFI) of positively-stained cells was measured, and the MFI of the
background (regions where no cells were detected) was
subtracted from this value.
Toxicity procedurerhAbs were added to cell cultures as described above.
Human astrocytes were incubated for 6 h prior to heatshock or remaining at 37 °C for 1.5 h. Cells were incubated with 6 μM Caspase-3/7 Green Detection Reagent(Invitrogen C10423) for the last 30 min of the incubation.
Cells were fixed with 4% PFA then underwent immunocytochemistry with the following antibodies: Mouse antiG3BP1 (1:100 BD Biosciences 611126), Goat anti-Humanconj AlexaFluor 568 (1:1000 ThermoFisher A-21090),
Goat anti-Mouse conj AlexaFluor 647. Coverslips were
mounted and visualized with a Nikon CSU-W1 spinningdisk confocal microscope.
Statistical analyses

Analyses were performed with GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA).
One-way ANOVA was used to compare flow cytometry and CyTOF data, unless there were less than threegroups in which case a Student’s T-test was appropriate.
For immune repertoire data additional corrections were
performed for multiple comparisons of frequencies [39].
Frequencies subjected to arcsine transformation andnon-parametric ANOVA was used with a post-hoc analysis (the Dunnett multiple comparison method) for pairwise comparison of patient groups with healthy donors.

Smith et al. Journal of Neuroinflammation(2024) 21:161

Results
Patient plasmablast frequency correlates with age

Adult patients presenting with ATM symptoms demonstrate an increased frequency of ­CD19+/lowCD27HI
plasmablasts in the blood compared to controls [30].
We hypothesized that pediatric patients presenting with
ATM symptoms would also demonstrate an increased
frequency of plasmablasts in the blood. To investigate

Page 5 of 16

this, we used conventional flow cytometry to identify(Fig. 1A) and calculate the frequency of plasmablasts inthe blood of 5 pediatric ATM patients and 10 pediatrichealthy controls (HC). While the frequency of plasmablasts in the blood of the pediatric ATM patients and pediatric HC was no different (2.1% vs 2.5%, p = NS) (data notshown), we did note that the frequency of plasmablasts inthe blood of pediatric and adult ATM patients increases

Fig. 1 Profiling of B cell subsets. A Gating strategy to obtain the frequency of plasmablast (PB) in each sample using traditional flow cytometry.
B Plasmablast frequency correlation with age in patients with acute transverse myelitis using traditional flow cytometry. C Plasmablast frequency
correlation with age in healthy controls using traditional flow cytometry. D–G Plasmablast subset frequencies in pediatric acute transverse myelitis
and healthy controls using CyTOF data. H, I TSNE plots of CyTOF data identifying 20 distinct B cell subsets in the cohort

Smith et al. Journal of Neuroinflammation(2024) 21:161

with age (Fig. 1B) whereas plasmablasts in the blood ofpediatric and adult HC does not (Fig. 1C).
Pediatric patient plasmablast frequency is reduced
compared to HC

To further our understanding of this early immune profiling data, we subjected the pediatric cohort (5 TM
patients and 10 HC) to CyTOF technology which provided frequency information on 15 B cell subsets using
a traditional gating strategy (Supplemental Fig. 1A and
Supplemental Table 1). This data demonstrates that theoverall frequency of plasmablasts in pediatric HC is significantly higher compared to pediatric ATM patients(p = 0.0007; Fig. 1D). Class-switched plasmablasts constituted the majority of the plasmablast population inpediatric HC (p = 0.002; Fig. 1E), while the frequency
of IgM + plasmablasts was higher in pediatric ATM(Fig. 1F), resulting in a switched:unswitched plasmablast ratio that was higher in the pediatric HC comparedto ATM (Fig. 1G). Next, we used FlowSOM analysis on
the OMIQ platform comparing pediatric ATM and HC
cohorts using the C­ D3−CD14− live singlets (Supplemental Fig. 1B) as the input to the FlowSOM clustering
which generated 20 unique B cell clusters in the dataset(Fig. 1H, I). Of those, Cluster 12 ­(CD27+CD38+ plasmablasts) displayed discordant frequency between the ATM
and HC cohorts, confirming our findings by traditional
gating of the dataset. Cluster 13 ­(IgM+CD22+) was also
noted to display discordant frequency between the ATM
and HC cohorts.
Pediatric patient serum IgG displays autoreactivity

Plasmablasts, and more specifically, class-switched
plasmablasts were not expanded in the pediatric ATM
patients, but the antibodies produced by this reduced
fraction of plasmablasts may be enriched for autoreactivity. To address this, we asked whether serum IgG fromthese patients displayed binding to neurons or glia inspinal cord tissue. We tested for binding on mouse spinal
cord since transverse myelitis symptoms originate withpathology in this tissue. We used spinal cord from wild
type (WT) mice with no evidence of neuroinflammation (Fig. 2A and Supplementary Fig. 2) and spinal cordfrom mice that had experienced hind limb paralysis due
to neuroinflammation following induction of Experimental Autoimmune Encephalitis (EAE) (Fig. 2B and Supplementary Fig. 2). Using wild type spinal cord, we did notobserve discordance in mean MFI binding to neurons or
astrocytes between serum IgG from the pediatric ATM
and HC groups (Fig. 2C). Serum IgG samples from thesegroups also demonstrated similar mean MFI binding inneurons of EAE spinal cord (Fig. 2D). However, the mean
MFI binding of astrocytes by pediatric ATM serum IgG

Page 6 of 16

was significantly reduced in EAE spinal cord compared tomean MFI astrocyte binding by pediatric HC serum IgG(Mean MFI 731 vs 275, p = 0.039) (Fig. 2D).
Pediatric patient plasmablast discordance in heavy chain
family use

The observations of reduced plasmablast frequency andreduced mean MFI binding to astrocytes in EAE spinal
cord by serum IgG of these pediatric ATM patients compared to pediatric HC prompted us to examine potential discordance in antibody gene distribution between
pediatric ATM and HC. To do this, we sorted plasmablasts from the blood of pediatric ATM patients and HC,then used our single cell PCR pipeline to obtain the frequency of heavy chain family usage among the plasmablast populations of each subject. Adult ATM patientsdisplay a robust expansion of VH4 family use compared
to controls [30], but we did not observe enrichment of
VH4 family use in the pediatric ATM patients comparedto pediatric HC (Table 2). VH3 family use in the pediatric ATM patients was significantly reduced in comparison to pediatric HC (p = 0.016), but this did not alter the
VH4:VH3 ratio of pediatric ATM patients compared to
HC. The plasmablasts from pediatric ATM patients did
display a higher mutation accumulation in comparison topediatric HC (p = 0.047), but this was not specific to plasmablasts from pediatric ATM patients expressing VH4
family genes.
Low prevalence of neuron binding to EAE spinal cord
by VH3 + and VH4 + antibodies from pediatric subjects

To further our understanding of whether plasmablast
immunogenetics influence binding properties of the antibodies they produce, we cloned antibodies expressedby 20 plasmablasts from the cohort in 4 categories:
VH3 + with no mutations (VH3 + SHM−), VH3 + withhigh mutation accumulation (VH3 + SHM +), VH4 + withno mutations (VH4 + SHM−) and VH4 + with highmutation accumulation (VH4 + SHM +) (Supplemental Table 2). We cloned and expressed these antibodies
as recombinant human antibodies (rhAbs) as previously
described [29, 39] to determine reactivity. In each category, 2 rhAbs originated with pediatric HC and 3 rhAbs
originated with pediatric ATM patients for a total of 20
rhAbs. We tested for binding on mouse spinal cord sincetransverse myelitis symptoms originate with pathology
in this tissue. We used spinal cord from wild type (WT)
mice with no evidence of neuroinflammation and spinal
cord from mice that had experienced hind limb paralysis due to neuroinflammation associated with inductionof Experimental Autoimmune Encephalitis (EAE). Of
the 20 rhAbs tested, one bound neurons in WT spinal
cord, 4 bound neurons in EAE spinal cord and one boundSmith et al. Journal of Neuroinflammation(2024) 21:161

Page 7 of 16

Fig. 2 IgG purified from serum samples bind neurons and astrocytes in mouse spinal cord. A, B Representative images from the lumbar spinal cordof WT (A) and EAE (B) mice. Green: IgG staining. Red: GFAP. Blue: MAP2. In the merge panels, yellow indicates co-stain of GFAP with the rhAb. The
MFI of astrocytic staining is indicated in parentheses. Scale bar: 20 μm. C, D Scatter plots of the mean fluorescent intensity of neuron and astrocyte
binding in WT (C) and EAE (D) tissue using IgG purified from patient and control serum. The average MFI of each cohort is provided below the x-axis

neurons in both WT and EAE spinal cord (Supplemental Table 2). Thus, 6 of the 20 rhAbs (30%) bound neurons in WT or EAE spinal cord. Of note, the two rhAbs
that bound neurons in WT spinal cord were VH3+, while

three of the five rhAbs that bound neurons in EAE spinal
cord used VH4 antibody heavy chain genes. Both pediatric ATM and HC subjects had PBs producing neuronbinding rhAbs.

Smith et al. Journal of Neuroinflammation(2024) 21:161

Page 8 of 16

Table 2 Antibody heavy chain profileHC

ATM

P-value

Percent VH4

26.02 ± 11.62

30.76 ± 12.74

NS

Percent VH3

73.24 ± 11.79

49.87 ± 9.9

P = 0.016

VH4:VH3

0.39 ± 0.24

0.68 ± 0.36

NS

Percent highly
mutated plasma‑
blasts9%23%

P = 0.047

Percent highly
17%
mutated VH4 + plas‑mablasts

22%

NS

Somatic hypermutation enhances glia binding to EAEspinal cord by VH3 + and VH4+ antibodies from pediatric
ATM subjects

In contrast to the low frequency of neuron-bindingrhAbs, we observed that 11 of the 20 rhAbs (55%) boundastrocytes in WT or EAE spinal cord (Fig. 3, Supplemental Fig. 3 and 4). All 5 of the EAE spinal cord neuron-binding rhAbs also bound astrocytes in EAE spinal
cord (Supplemental Table 2). An additional 6 rhAbs
acquired astrocyte binding in EAE spinal cord, but did
not bind neurons in EAE spinal cord. Figures 3A, B display PTM-6 rhAb as an example of binding astrocytes
in both WT and EAE spinal cord and PTM-12 rhAb as
an example of binding astrocytes in EAE spinal cord
only. Of the 11 that bound astrocytes in EAE spinal cord,
4 used VH3 family genes and 7 used VH4 family genes(Fig. 3C). Astrocyte binding intensity in EAE spinal cord
was dependent on somatic hypermutation accumulationin the pediatric ATM cohort (Fig. 3D), since antibodies
with somatic hypermutations bound astrocytes in EAE
spinal cord with significantly higher mean MFI (2.5-fold)than antibodies without SHM (565 v 1407, p = 0.0008).
The pediatric HC cohort had similar mean MFI binding
to astrocytes in EAE spinal cord, independent of SHM
accumulation.
To determine if binding was specific to astrocytes inspinal cord, we also investigated binding of the rhAbs
to astrocytes in WT and EAE mouse brain (Fig. 4,
Supplemental Table 2, and Supplemental Figs. 5 and
6). Of the 11 that bound astrocytes in EAE spinal
cord, 9 bound astrocytes in EAE brain. Two of the
11 that bound astrocytes in EAE spinal cord did notbind astrocytes in EAE brain and one acquired astrocyte binding to EAE brain but did not bind astrocytes
in EAE spinal cord. Figures 4A, B display the PTM-6
and PTM-12 rhAbs as examples of binding astrocytes
in both WT and EAE brain. Thus, 5 of the 20 rhAbs(25%) displayed astrocyte binding in WT brain and 12of the 20 rhAbs (60%) displayed astrocyte binding in

EAE brain (Fig. 4C). Astrocyte binding in EAE brain
by rhAbs was highly skewed towards those that originated from plasmablasts of pediatric HC (8 of 8 in
HC compared to 4 of 12 in ATM). Astrocyte binding
strength by rhAbs from pediatric HC in EAE brain was
independent of SHM accumulation as we observed for
EAE spinal cord (data not shown).
Primary human astrocyte binding is similar in pediatric
ATM patients and controls

Next, we investigated the binding properties of the 20
rhAbs to primary human astrocytes (Fig. 5, Supplemental Fig. 7). Of the 20 rhAbs tested, 8 bound to humanprimary astrocytes (Fig. 5A). Five of the 8 rhAbs thatbound primary human astrocytes were from the pediatric ATM patients and the remaining 3 were from pediatric HC. Primary human astrocyte binding was observedwith both VH3 + rhAbs and VH4 + rhAbs independentof SHM accumulation. The mean MFI of human primary astrocyte binding by rhAbs from the pediatric ATM
cohort was similar to the mean MFI of human primary
astrocyte binding by rhAbs from the pediatric HC cohort(Fig. 5B). No general immunogenetic features (including VH gene use, JH gene use, CDR3 length or charge)of the 5 rhAbs from pediatric ATM patients that boundprimary human astrocytes displayed commonalities thatmight infer preference for astrocyte binding (Table 3).
However, we did observe 3 motifs in FR2 and FR3 shared
among rhAbs from either pediatric ATM or HC thatbound human primary astrocytes (Fig. 5C). We also performed immunocytochemistry on the human epithelial
cell line Hep-2 (Supplemental Fig. 8), a test commonly
employed to detect anti-nuclear antibodies in SLE anda cell line expressing AQP4 (Supplemental Fig. 9), which
identifies subjects that may be diagnosed with Neuromyelitis Optica. None of the rhabs in this cohort was reactive to AQP4 + HEK293 cells or Hep-2 cells.
Astrocyte binding rhAbs from pediatric ATM patientsenhance stress granule formation, but are not toxic
to astrocytes

To investigate whether astrocyte binding impacts astrocyte health, we first determined the optimal time ofrhAb internalization (Supplemental Fig. 10) in primary
human astrocytes. Then the cultured primary humanastrocytes were incubated with two of the rhAbs that
displayed the highest intensity of human astrocyte binding (PTM-6 and PTM-12; Fig. 5B) and one rhAb that
does not bind astrocytes as a negative control (PTM-8).
Following this incubation, the percentage of cells with
­G3BP1+ puncta was measured as an indication of astrocyte stress, and the percentage of Caspase 3/7 + cells wasmeasured as an indication of astrocyte toxicity (Fig. 6).

Smith et al. Journal of Neuroinflammation(2024) 21:161

Page 9 of 16

Fig. 3 Plasmablast rhAbs bind neurons and astrocytes in mouse spinal cord. A, B Representative images of rhAbs binding neurons and astrocytes
in the lumbar spinal cord of WT (A) and EAE (B) mice. Green: IgG staining. Red: GFAP. Blue: MAP2. In the merge panels, yellow indicates co-stainof GFAP with the rhAb. The MFI of astrocytic staining is indicated in parentheses. Scale bar: 20 μm. C Pie charts summarizing the number of rhAbs
binding astrocytes in WT and EAE spinal cord tissue. D Scatter plots of the mean fluorescent intensity of the 11 astrocyte-binding rhAbs in EAE
spinal cord tissue where the rhAbs are binned according to cohort (ALL, HC or ATM) and somatic hypermutation (SHM) accumulation statusof the antibody heavy chain where “SHM−” indicates there is no SHM in the rhAb heavy chain and “SHM + ” indicates there is SHM in the rhAb heavy
chain. Horizontal dashed line indicates the threshold of positivity which is set at 366.3. The average MFI of each cohort is provided below the x-axis

We used heatshock as a known inducer of G3BP1 punctate formation to provide a threshold of expected astrocyte stress [45]. Indeed, primary human astrocytes

incubated with the negative control rhAb displayed a2.5-fold increase of ­G3BP1+ puncta following heatshock(p = 0.012) (Fig. 6C). Incubation of human astrocytes

Smith et al. Journal of Neuroinflammation(2024) 21:161

Page 10 of 16

Fig. 4 Plasmablast rhAbs bind neurons and astrocytes in mouse brain. A, B Representative images of rhAbs binding neurons and astrocytes
in the brain of WT (A) and EAE (B) mice. Green: IgG staining. Red: GFAP. Violet: MAP2. Blue: DAPI. In the merge panels, yellow indicates co-stainof GFAP with the rhAb. The MFI of astrocytic staining is indicated in parentheses. Scale bar: 20 μm. C Pie charts summarizing the number of rhAbs
binding astrocytes in WT and EAE spinal cord tissue. Positive binding rhAbs are binned according to antibody variable heavy chain use (VH3+or VH4+)

with PTM-6 rhAb resulted in a sixfold greater frequency
of cells with ­G3BP1+ puncta than the negative control
rhAb (p = 0.001) (Fig. 6C). Incubation of human astrocytes with PTM-12 rhAb resulted in a fourfold greater
frequency of cells with ­G3BP1+ puncta than the control(p = 0.001) (Fig. 6C). Heatshock treatment did not further increase the frequency of G3BP1 + puncta exposed
to PTM-6 or PTM-12 (Fig. 6C). While both PTM-6 andPTM-12 induced astrocytic stress, neither rhAb induced
astrocytic toxicity as measured by the frequency of Caspase 3/7 + cells (Fig. 6D).

Discussion
Patients of any age experiencing initial acute transverse
myelitis symptoms are at a critical point when intervention could impact prognosis [23, 37]. For example, use of
immunosuppressive therapeutics such as corticosteroids
and plasma exchange have shown efficacy in the adult
ATM population [48]. However, the use of immunotherapy in the pediatric population could introduce additional
issues with extended use. For example, prolonged useof corticosteroids can lead to bone, skin and endocrinecomplications particularly in pediatric populations [5].
Thus, a deeper understanding of the biology at these earlystages of disease in the pediatric ATM population could
reveal which pediatric ATM cases would benefit from
immunosuppressive therapeutics. We previously demonstrated that adult ATM patients display an expansion of
VH4 + PB in the blood, producing antibodies that bind toneurons. The initial focus here was to determine if thesesame features are displayed in pediatric ATM patients.
We began our profiling of pediatric ATM by comparing
the frequency of peripheral B cell subsets in pediatric
ATM and HC (Fig. 1). In this analysis, we observed thatthe frequency of PB was significantly reduced in the pediatric ATM cohort compared to pediatric HC (p = 0.0007).
One explanation for this observation is that PBs in pediatric ATM cases are not readily detected in the blood
because they have extravasated to the central nervous
system, presumably in response to inflammation in thiscompartment. However, we further observed that the
total PB population in pediatric ATM was enriched for
IgM + PB resulting in a ratio of switched:unswitched PB
that was significantly higher in pediatric HC compared topediatric ATM. This finding was further confirmed using
FlowSOM analysis. Thus, the frequency of class-switched
PBs in particular is reduced in the blood of pediatricATM. Others have demonstrated that class-switchedSmith et al. Journal of Neuroinflammation(2024) 21:161

Page 11 of 16

Fig. 5 Plasmablast rhAbs bind to human primary astrocytes. A Representative images for the 8 rhAbs that bind primary human astrocytes. Green:
rhAb staining. Red: GFAP. Blue: DAPI. In the merge panels, yellow indicates co-stain of GFAP with the rhAb. The MFI of astrocytic staining is indicatedin parentheses. Scale bar: 20 μm. B Scatterplots of the MFI of human primary astrocytes where the rhAbs with positive binding are binned
according to cohort (HC or ATM). The average MFI of each cohort is provided to the left of the x-axis. C Alignment of the antibody heavy chain
sequences using Clustal Omega (EMBL-EBI). Only those sequences of rhAbs with positive binding to human primary astrocytes are shown and arebinned according to cohort (ATM above and HC below). Green boxes demarcate the 3 motifs shared between astrocyte binding rhAbs. One lettercodons that underwent replacement mutation are indicated in red letters. Codon positions which have a fully conserved residue in all sequences
are indicated by “*”. Conservation between groups of strongly similar properties are indicated by “:”. Conservation between groups of weakly similar
properties are indicated by “.”(IgA+) PB suppress CNS inflammation in mouse models of MS [40], which may support the notion that suppression of class-switched subsets allows for the CNS
inflammation displayed in pediatric ATM patients. However, none of these pediatric ATM cases advanced to MS
diagnosis within the 2 years of this study, despite reduced
frequency of class-switched PBs. Future investigations

should thus focus on the contribution of PB subsets(IgM + versus class-switched) and longitudinal profilingin pediatric ATM which is likely crucial to understanding
the potential cause of a first (and perhaps only) ATM episode versus conversion to life-long autoimmune disease(expected 22%) [8, 18].

Smith et al. Journal of Neuroinflammation(2024) 21:161

Page 12 of 16

Table 3 Gene information for primary human astrocyte positive rhAbs
rhAb namerhAb category

VH geneJH gene

CDR3
AA length

CDR3chargeVH#RM

VL geneJL geneVL#RM

Patient

PTM-2

VH3 + SHM−

VH3-7
JH6

25

0

0

VK1-39
JK5

0

PTM-A

PTM-4

VH3 + SHM+

VH3-7
JH4

10

0

11

VK2-30
JK4

2

PTM-A

PTM-6

VH3 + SHM+

VH3-30
JH4

20

1

9

VK1-27
JK5

4

PTM-B

PTM-9

VH4 + SHM−

VH4-39
JH4

19

1

0

VK3-20
JK2

0

PTM-B

PTM-12

VH4 + SHM+

VH4-4
JH4

14

−3

5

VK3-11
JK2

9

PTM-C

VH variable heavy, JH junction heavy, CDR3 complementarity determining region 3, AA amino acid, #RM number of replacement mutations, VL variable light

Fig. 6 Astrocyte binding rhAbs induce astrocyte stress, but not apoptosis. A Representative images of untreated and heatshock treated humanprimary astrocytes to demonstrate G3BP1 + puncta formation. White arrowheads indicate G3BP1 + puncta. B Representative images of primary
human astrocytes incubated with PTM-6 or PTM-12 rhAbs showing induction of G3BP1 + puncta. Green: rhAb. Red: G3BP1. Blue: DAPI. Violet: Active
caspase 3/7. In the merge panels, yellow indicates co-stain of G3BP1 with the rhAb. Scale bar: 20 μm. C Summary statistics for G3BP1 puncta + cells
as an indication of stress. D Summary statistics for Caspase 3/7 + cells as an indication of apoptosis

We next sought to investigate whether this reduced PB
population in pediatric ATM maintained its capacity to
produce autoreactive immunoglobulin. To address this,

we investigated the binding potential of purified IgG fromserum samples (Fig. 2) and rhAbs cloned from individual
PBs (Figs. 3, 4, 5). We had expected to observe substantial

Smith et al. Journal of Neuroinflammation(2024) 21:161

neuronal binding by purified IgG and rhAbs from pediatric ATM patients compared to pediatric HC as an extension of our adult ATM patient data observations. Instead,these two populations displayed similar frequencies andbinding intensities to neurons in all tissue types tested.
However, in EAE spinal cord tissue, we observed that
purified IgG from pediatric HC had an increased mean
MFI of binding to astrocytes compared to purified IgGfrom pediatric ATM. Astrocyte binding was also similarin these two populations in WT tissue samples. To examine this at the single cell level, we cloned 20 antibodies
expressed by single PB from the cohort and found that
11 of them bound to astrocytes in EAE spinal cord. The 3
with the highest MFI were from pediatric ATM patientswith high accumulation of SHM. A similar observation
was made with EAE brain tissue with an enrichment ofastrocyte binding by rhAbs that had high accumulationof SHM. While astrocyte reactivity of most of the rhAbs
was confirmed in human primary astrocytes (Fig. 5), we
did not observe any features of immunogenetics thatinferred binding as both VH3 + and VH4 + rhAbs boundhuman primary astrocytes independent of SHM accumulation. Also, while the 5 rhAbs from the pediatric
ATM PBs bound to astrocytes with a similar cytoplasmic pattern, one of the 3 astrocyte binding rhAbs fromthe pediatric HC cohort (PHC-5) bound exclusively inthe nucleus of human primary astrocytes, not the cytoplasm. The PHC-5 rhAb and purified IgG from this same
pediatric HC subject displayed typical astrocytic binding in the EAE spinal cord and brain tissue. Of note, thePHC-5 rhAb contains the 3 codon motifs associated withastrocyte binding by 5 of the pediatric TM rhAbs andthe other 2 pediatric HC rhAbs, suggesting that otherimmunogenetic features may contribute to binding of
PHC-5 rhAbs to the specific antigen target located in
the nucleus. Curiously, none of these three motifs werein codon positions that had undergone SHM but wereinstead, original to the germline configuration in conserved framework regions of the variable domain. Future
investigations should focus on (1) elucidating the impactof these 3 motifs on astrocyte binding properties, and (2)identifying the antigen targets as the binding patterns ofthese astrocyte-binding rhAbs are distinct.
The PTM-6 and PTM-12 rhAbs displayed the highest
MFI of the human primary astrocyte binders (Fig. 5). To
address the impact of these rhAbs on astrocyte function,
we incubated human primary astrocytes with these two
rhAbs and calculated the frequency of human primary
astrocytes with G3BP1-marked cytoplasmic puncta
as an indication of astrocytic stress [16], and Caspase
3/7 + human primary astrocytes as an indication of apoptotic events (Fig. 6) [45]. Our results indicated that incubation with PTM-6 and PTM-12 alone induced astrocytic

Page 13 of 16

stress but did not advance the human primary astrocytes
to apoptosis. Subjecting the human primary astrocytes
to a heatshock-induced stress event following incubation with PTM-6 did not further exacerbate astrocytic
stress and did not advance the human primary astrocytes
to apoptosis. Of note, PTM-6 displayed a higher MFI of
human astrocyte binding than PTM-12, which may havefacilitated its ability to induce a greater astrocytic stressresponse compared to PTM-12. A comparison of antibodies derived from pediatric ATM subjects to thosederived from HC subjects (such as PHC-5 and PHC-6)
may clarify whether induction of stress is common to thecodon motifs associated with astrocyte binding, or distinct to pediatric ATM subjects.
Stress granules are membrane-less organelles that
assemble during stress, injury, and neurological disease[20, 42, 47]. They consist of stalled mRNA transcripts,
translation factors, ribosomal subunits, RNA-binding
proteins, as well as G3BP1 which is a core component
and required for their assembly [53]. While they arethought to be an adaptive response to unfavorable conditions and are normally cleared upon resolution ofstress, their chronic presence is cytotoxic in certain cell
types [55]. Exposure to IgG against a particular target can
induce stress granules in neurons resulting in death [27],but this is the first report demonstrating that exposure
to IgG can induce stress granules in astrocytes, but does
not lead to death. In future studies, it would be valuable
to determine whether chronic exposure to astrocytebinding antibodies is cytotoxic, or whether presenceof complement is required to induce cytotoxicity [32].
Examination of other mechanistic pathways in live-cellincubation assays will also determine the pathogenicity ofastrocyte-binding antibodies [21].
The impact of this stress response by astrocytes mediated by astrocyte-binding antibodies on CNS healthremains unknown. Astrocytes are essential to BBB integrity [25] and outnumber neurons 50:1 [50]. Astrocytes
also transport nutrients to neurons and oligodendrocytes, and astrocyte activation after antibody binding [10]can induce production of cytotoxic factors and enhance
immune responses, ultimately forming glial scars that
impair remyelination and axonal regeneration [12, 26].
Astrocyte release of inflammatory cytokines disrupts the
BBB in MS by activating endothelial cells [1, 34], so astrocyte-binding autoantibodies could challenge BBB integrity and affect entire regions of neurons as observed in
NMO with autoantibodies against AQP4 [41, 54]. Given
these potentially deleterious effects of astrocyte-binding antibodies on astrocyte function, it stands to reason
that it would be beneficial to suppress PB expansion inpediatric ATM patients experiencing CNS inflammation to reduce further injury caused by astrocyte-bindingSmith et al. Journal of Neuroinflammation(2024) 21:161

antibodies that PBs from pediatric ATM patients could
potentially produce.
However, this conclusion does not resolve the observation that pediatric HC subjects display increased astrocyte binding of purified IgG from serum, and antibodies
expressed by individual PB from pediatric HC subjects
also bind astrocytes. It is possible that this dichotomy
may relate to emerging data suggesting that astrocyte
engagement is an important mechanism to reduce neuroinflammation and initiate/sustain neuroprotection [11,33]. This concept was originally established by observations that ischemic preconditioning (IPC) promotes neuroprotection from stroke through mechanisms involving
astrocyte activation, including mild responses thatwould be deleterious to cellular health if larger in magnitude [24]. For example, astrocyte release of nuclear
factor erythroid-derived 2-related factor 2 (Nrf2) following preconditioning resulted in neuroprotection fromneuroinflammation associated with stroke [17]. Astrocyte activation through the P2X7 receptor has also beenstrongly implicated in neuroprotection from stroke [19]and astrocyte activation is important in recovery fromtraumatic brain injury [36]. In the context of Multiple
Sclerosis, astrocyte activation through Oncostatin M
release activates a feed-forward loop that reduces disease
severity in a mouse model of MS [51]. Astrocytes canalso modulate both acute and progressive mouse modelsof MS through a mechanism involving PD-L1 [31].
None of these previous studies investigated the impactof astrocyte-binding antibodies on astrocyte activation.
We present evidence here that exposure of astrocytes
to astrocyte-binding rhAbs induces astrocytic stress,
but not astrocytic death. This raises the intriguing possibility that astrocyte-binding rhAbs may induce a stressresponse similar to the paradigm of ischemic preconditioning as a pathway to neuroprotection. This study islimited in that (1) we did not have access to cells in the
CSF for further study of CNS autoimmunity in pediatric cases, (2) the impact of multiple vaccinations andincreased frequency of viral illnesses during childhood
is not considered and (3) antigen targets were not identified. Nevertheless, our observations of increased plasmablast frequencies in pediatric HC, increased titers ofserum-derived IgG from pediatric HC that bind astrocytes, and increased astrocyte reactivity by rhAbs in thepediatric HC population without the need for SHM todrive astrocyte reactivity all support the concept thatastrocyte-binding rhAbs may promote a neuroprotective astrocyte population at early stages of life. Indeed,
the differences between pediatric ATM and adult ATM B
cell repertoires may suggest that the two immune mediated spinal cord pathologies have distinct pathogenensis

Page 14 of 16

and should be studied separately. Such subjects would beprotected from clinical manifestations of neuroinflammatory disease, whereas those with a less robust astrocyte-focused humoral immune response (as observedin our pediatric ATM patients using purified IgG pools)
would manifest neuroinflammatory disease. To ourknowledge, none of the current immunotherapies usedin the treatment of pediatric ATM are designed to specifically modulate this potential mechanism. Further
investigations of the impact astrocyte-binding antibodies have on astrocyte activation would further elucidate
this potential mechanism of neuroprotection and leadto novel therapies specifically beneficial to the pediatricpopulation.

Conclusions
Pediatric ATM patients display reduced frequency of
peripheral PB and reduced titer of astrocyte-binding IgG
purified from serum samples compared to pediatric HC.
Nevertheless, astrocyte-binding antibodies expressed by
PBs identified from pediatric ATM patients induce anastrocyte stress response, but not astrocytic apoptosis.
The downstream impact of this astrocytic response has
not been elucidated, but introduces an intriguing possibility of antibody-mediated neuroprotection.
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03127-2.
Supplementary Material 1.
Supplementary Material 2.
Acknowledgments
We would like to thank the patients and healthy subjects who participated inthese studies, as well as their parents, relatives, and the clinicians for making
this work possible. Special thanks to Denny and Sandy Miller for use of theirretreat home to write this paper. This manuscript is dedicated to the memoryof Sara J. Ireland (d. 9.3.2022), our beloved colleague and friend who intro‑
duced us to the world of advanced immune profile platforms for this paper.
Author contributions
CS, KielMT, SGMP, YLM, WZ, KeyMT, JRR, CJ, DSS, AW, RT, SC, and YQ partici‑
pated in data generation; CS, KielMT, WZ, KeyMT, YQ and NLM prepared thefigures, CS, SGMP, LGC, RHS, AMS, LN, BMG and NLM edited the manuscript. All
authors reviewed the manuscript.
Funding
REDCap was used to archive clinical data associated with samples and was
funded by NIH to UTSW (Grant UL1 TR003163). This manuscript was funded by
grants awarded to Dr. Nancy Monson from the Transverse Myelitis Foundationand the National Institutes of Health (NS098229, NS102417 and NS123398). KT
was supported by NIH grant K22 NS123508.
Data and materials availability
Data are available upon request to the corresponding author.

Smith et al. Journal of Neuroinflammation(2024) 21:161

Declarations
Ethics approval and consent to participate
All subjects and/or their legally authorized study partners signed the written
informed consent approved by the Institutional Review Board of the UTSouthwestern Medical Center (UTSW), in accordance with the Federal-wide
Assurance on file with the Department of Health and Human Services (USA).
Competing interests
The authors state no competing interests relative to this manuscript.
Author details
1
UT Southwestern Department of Neurology, Dallas, TX, USA. 2 The Brain
Tumor Translational Laboratory, Department of Cell Biology and Physiology,
University of New Mexico Health Sciences Center, Albuquerque, NM, USA.
3
University of New Mexico Comprehensive Cancer Center, Albuquerque, NM,
USA. 4 UT Southwestern O’Donnell School of Public Health, Dallas, TX, USA. 5 J.
Craig Venter Institute, La Jolla, CA, USA. 6 National Library of Medicine, National
Institutes of Health, Bethesda, MD, USA. 7 Department of Neurology, Universityof Kentucky, Lexington, KY, USA. 8 UT Southwestern Department of Immunol‑
ogy, Dallas, TX, USA.
Received: 11 March 2024 Accepted: 8 May 2024

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