STLV-1 Commonly Targets Neurons in the Brain of Asymptomatic Non-Human Primates

Abstract

The human T-cell leukemia virus (HTLV)-1 is responsible for an aggressive neurodegenerative disease (HAM/TSP) and multiple neurological alterations. The capacity of HTLV-1 to infect central nervous system (CNS) resident cells, together with the neuroimmune-driven response, has not been well-established. Here, we combined the use of human induced pluripotent stem cells (hiPSC) and of naturally STLV-1-infected nonhuman primates (NHP) as models with which to investigate HTLV-1 neurotropism. Hence, neuronal cells obtained after hiPSC differentiation in neural polycultures were the main cell population infected by HTLV-1. Further, we report the infection of neurons with STLV-1 in spinal cord regions as well as in brain cortical and cerebellar sections of postmortem NHP. Additionally, reactive microglial cells were found in infected areas, suggesting an immune antiviral response. These results emphasize the need to develop new efficient models by which to understand HTLV-1 neuroinfection and suggest an alternative mechanism that leads to HAM/TSP.

Full text

STLV-1 Commonly Targets Neurons in the Brain of
Asymptomatic Non-Human Primates
Brenda Rocamonde,a, g Sandrine Alais,a, g Rodolphe Pelissier,bValerie Moulin,cBrigitte Rimbaud,cRomain Lacoste,c
Noemie Aurine,dCamille Baquerre,dBertrand Pain,dYuetsu Tanaka,eCyrille Mathieu,fHélène Dutartrea, g
a
Centre International de Recherche en Infectiologie, équipe d’Oncogenèse Rétrovirale, INSERM U1111 - Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole
Normale Supérieure de Lyon, Université Lyon, Lyon, France
b
Centre International de Recherche en Infectiologie, équipe Immunobiologie des Infections Virales, INSERM U1111 - Université Claude Bernard Lyon 1, CNRS, UMR5308,
Ecole Normale Supérieure de Lyon, Université Lyon, Lyon, France
c
Station de Primatologie-UAR846-CNRS, France
d
Univ Lyon, Université Lyon 1, INSERM, INRAE, Stem Cell and Brain Research Institute, U1208, USC1361, Bron, France
e
Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Nishiharacho, Okinawa, Japan
f
Centre International de Recherche en Infectiologie équipe Neuro-Invasion, TROpism and VIRal Encephalitis, INSERM U1111 - Université Claude Bernard Lyon 1, CNRS,
UMR5308, Ecole Normale Supérieure de Lyon, Université Lyon, Lyon, France
g
Equipe labellisée par la Fondation pour la Recherche Médicale, Labex Ecofect
ABSTRACT The human T-cell leukemia virus (HTLV)-1 is responsible for an aggressive
neurodegenerative disease (HAM/TSP) and multiple neurological alterations. The capacity
of HTLV-1 to infect central nervous system (CNS) resident cells, together with the neuro-
immune-driven response, has not been well-established. Here, we combined the use of
human induced pluripotent stem cells (hiPSC) and of naturally STLV-1-infected nonhu-
man primates (NHP) as models with which to investigate HTLV-1 neurotropism. Hence,
neuronal cells obtained after hiPSC differentiation in neural polycultures were the main
cell population infected by HTLV-1. Further, we report the infection of neurons with
STLV-1 in spinal cord regions as well as in brain cortical and cerebellar sections of post-
mortem NHP. Additionally, reactive microglial cells were found in infected areas, sug-
gesting an immune antiviral response. These results emphasize the need to develop
new efficient models by which to understand HTLV-1 neuroinfection and suggest an al-
ternative mechanism that leads to HAM/TSP.
KEYWORDS HTLV-1, neurotropism, inflammation, microglial response
The human T-cell leukemia virus (HLTV)-1 affects 10 to 20 million people worldwide.
After a long asymptomatic phase (20 to 30 years), between 1 and 5% of HTLV-1-infected
subjects will develop a neurodegenerative disease known as HTLV-1-associated myelopa-
thy/tropical spastic paraparesis (HAM/TSP) (1). HAM/TSP is manifested as an ensemble of
motor dysfunctions that evolve toward the paralysis of lower limbs, a consequence of an
immune-mediated demyelinated thoracic cord (2). Corticoid therapy is unable to stop the
progression of the disease, and no effective treatment has been developed due to the poor
comprehension of the mechanisms initiating demyelination. A sustained, exacerbated
inflammation triggered by infiltrated lymphocytes has been proposed as the main mecha-
nism (3). Inflammatory and HTLV-1-specificCD8
1
T-cell lymphocytes, both infiltrated and
clonally expanded, can be found in the spinal cord sections (4) and Cerebrospinal Fluid
(CSF) of HAM/TSP postmortem samples (5), suggesting a CSF-compartmentalized antigen-
driven immune response (presumably HTLV-1 specific) at later stages of the disease. In vitro
experiments have shown the susceptibility of microglia, astrocytes, and neurons (originated
from both tumoral cell lines and primary cells) to HTLV-1 infection (6–9), and HTLV-1 RNA
was found in spinal cord astrocytes via the in situ hybridization of the postmortem tissues of
Invited Editor Steven Jacobson, National
Institutes of Health
Editor Diane E. Griffin, Johns Hopkins
Bloomberg School of Public Health
Copyright © 2023 Rocamonde et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Brenda
Rocamonde,
brenda.rocamonde@gmail.com, or Hélène
Dutartre, helene.dutartre@ens-lyon.fr.
The authors declare no conflict of interest.
Published 21 February 2023
March/April 2023 Volume 14 Issue 2 10.1128/mbio.03526-22 1
OPINION/HYPOTHESIS

HAM/TSPpatients(10).However,thein vivo infection of other neuronal populations remains
unclear (11), and it is not fully understood whether their infection is sufficient to initiate
T-cell-dependent chronic inflammation. Notably, investigating HTLV-1 neurotropism is a
challenging task due to (i) the absence of suitable in vitro models that can recapitulate the
complexity of the Central Nervous System (CNS) cytoarchitecture, (ii) the limited access to
myelinated postmortem samples from infected individuals, and (iii) the lack of tools that are
available to be used to investigate HTLV-1 infections (i.e., GFP-reportable viral particles),
compared to infections by other retroviruses. In contrast, naturally STLV-1-infected nonhu-
man primates (NHP) represent a valuable animal model for HLTV-1 infection, as the simian
homolog of HTLV-1 shares more than 99% of its genomic homology (12) and offers the pos-
sibility of accessing neural tissue before the demyelinating phase of the infection.
To get insights into HTLV-1 neurotropism, we first infected human induced pluripo-
tent stem cells (hiPSC)-derived neural cell polycultures with an HTLV-1-infected cell line
via co-cultivation. Then, we analyzed CNS samples from a cohort of nonhuman primates
that were naturally infected by STLV-1 to investigate HTLV-1 neural infection in vivo.This
unique model allowed us to investigate HTLV-1 neurotropism during the latent phase of
the disease, specifically, before the manifestation of neural inflammation. Hence, we
demonstrated STLV-1 neurotropism in vitro and detected, for the first time, the presence
of viral proteins in neurons from the spinal cord and cortical regions of STLV-1-infected
NHP. Strikingly, few microglia and astrocytes were positive for viral proteins. In contrast,
we found reactive microglia in close apposition to infected neurons, suggesting a local
immune response to STLV-1 infection that is potentially responsible for an early inflam-
matory response to the infection. Taken together, these findings raise new hypotheses
on the mechanisms that trigger the neural inflammation that was observed in HAM/TSP.
RESULTS
HTLV-1 preferentially infects neuronal cells in human iPSC-derived neural poly-
cultures. We used human iPSC-derived neural polycultures to investigate neuronal sus-
ceptibility to HTLV-1 infection in vitro. The productive infection of neural cells co-cultured
with C91-PL cells was monitored by the expression of the HTLV-1 Tax oncoprotein, as pre-
viously reported (13). Tax expression was detected mainly in neuronal cells expressing
beta-III-Tubulin (Tuj1) (Fig. 1A), suggesting the productive infection of the neuronal cells.
In these cells, Tax was mainly located in the perinuclear cytoplasm (Fig. 1B, arrowhead).
Infection was also associated with the modification of the cell morphology, especially with
the highest ratio of infected cells, with polynucleated cells and/or cells presenting frag-
mented nuclei suggesting a cytopathic effect (Fig. 1B, asterisks).
NHP were naturally infected with STLV-1 to model HTLV-1 neuroinfection in vivo.
Next, we addressed the question of neuronal susceptibility to HTLV-1 infection in vivo due
to its extremely high similarity to STLV-1. We took advantage of a cohort of naturally STLV-
1-infected NHP to investigate the viral neurodistribution. In this cohort, the average age of
NHP at the moment of the analysis was 23 years, with an unknown time of latent infection.
No motor dysfunction was reported by the animal keepers during the routine observation
of the animals, although no specific motor test was performed to address motor function-
ality. An overall low proviral load (PVL) was measured in blood periferal blood mononu-
clear cells (PBMC) (0.06 to 630 copies/10
5
cells) (Table 1). Strikingly, immunohistochemistry
in the spinal cord, cerebellum, and cortex revealed the presence of the viral Tax protein in
almost all of the animals (Fig. 2A; Table 1). The frequency of Tax
1
cells in the spinal cord-
infected regions were between 10% and 25% of the total cells analyzed, and no correlation
was identified with the PVL. The Tax protein was predominantly localized in the perinu-
clear cytoplasm, which is consistent with our in vitro observations and with previous
reports of Tax expression in in vitro infected primary cells or cell lines (8). Additionally, we
detected the concomitant cytoplasmic expression of the Gag p19 matrix protein of STLV-1
in the Tax
1
cells (Fig. 2B), suggesting the productive infection of the neural cells.
STLV-1 in the CNS of naturally infected NHP confirms neuronal tropism. HTLV-1
DNA was previously reported in the astrocytes of postmortem spinal cord samples
from a HAM/TSP patient (10). However, the HTLV-1 infection of other neural cell types
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was not investigated or reported. Thus, we addressed whether other CNS resident cell
types could be infected by STLV-1 in naturally infected NHP. As expected, the Tax pro-
tein was detected in 2% of the spinal cord astrocytes, and it was identified based on
their expression of GFAP, confirming their susceptibility to the infection, as in human
cases (Fig. 3A). Unexpectedly, microglial cells were also positive for Tax staining (10%
of Iba1
1
cells), whereas the oligodendrocyte lineage cells expressing the transcription
factor Olig2 were Tax negative (Fig. 3A). Consistent with our previous in vitro observa-
tions, the Tax protein was mainly localized in NeuN
1
neurons in the spinal cord as well
as in the cortical regions (Fig. 3B, up). In the cerebellum, staining was localized in the
ganglionic layer but not in NeuN
1
neurons. A few big neuronal cells from the Purkinje
cells/Golgi cells layer also expressed Tax (Fig. 3B, down). Such results confirm that
STLV-1 can reach at least the cortical and cerebellar regions. Overall, the frequency of
TABLE 1 List of naturally SLTV-1-infected and naive NHP, together with their PVL, and the
presence of Tax
1
cells in the spinal cord, cortex, and cerebellum
a
Number STLV-1 PVL (copies/10
5
PBMC) % cells Tax
+
spinal cord
Tax
+
Cerebellum Cortex
01 Neg na na na na
02 Neg na na na na
03 Pos 55 23.35 11
04 Pos 0.06 19.44 11
05 Pos 395 15.12 11
06 Pos 68 9.99 11
07 Pos 54 18.72 11
08 Pos nd 14.14 11
09 Pos 630 17.50 11
10 Pos 112 0 22
a
na, not assessed; nd, not determined.
FIG 1 HTLV-1 infects neural cells and triggers a glial response. (A) hiPSC-derived neural cell
polyculture cocultured with either JK or C91PL cells at two different ratios for five days. Scale bar:
100
m
m. (B) Tax
1
cells (arrowheads) in hiPSC presenting different morphologies, and polynucleated
cells (asterisks) expressing the Tuj1 neuronal marker. Scale bar: 20
m
m.
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Tax in the CNS resident cells showed that almost 80% were NeuN
1
neurons, 20% were
Iba1
1
microglial cells, and few were GFAP
1
astrocytes (Fig. 3C), indicating a specific
neuronal tropism of STLV-1.
STLV-1 is associated with a local neuroimmune response. Numerous microglial
cells (Iba1
1
) that were positive or not for the infection were frequently localized in
areas surrounding infected cells (Fig. 4A). Notably, in several spinal cord regions, micro-
glial cells were found in close apposition to Tax
1
cells (most likely neuronal cells).
Some of these surrounding microglial cells showed an amoeboid morphology, with
extended processes that contact Tax
1
cells (Fig. 4A, arrowheads). This microglial cell
phenotype is characteristic of activated phagocytic microglia. Moreover, the expression
of the CD40 marker (Fig. 4B, arrowheads), a reliable indicator of microglia activation,
confirmed the presence of reactive microglia in the infected areas.
DISCUSSION
To date, the mechanisms involving HTLV-1-driven neurodegeneration are still not
well-understood. The number of neurological affections triggered by HTLV-1 infection is
probably underestimated, and information regarding the cell-type specific neurotropism
is limited. Previous studies have reported the in vitro infection of astrocytes, microglia,
and neuronal monocultures (6–8). However, the frequency of infected cells and the dif-
ferential susceptibility among cell types due to the bystander effect of neighboring cells
have not been evaluated. Here, we showed that neurons are more susceptible to HTLV-1
infection than glial cells on both hiPSC-derived in vitro neural polycultures and postmor-
tem samples from NHP. Interestingly, the expression of the HTLV-1 Tax protein in our in
vitro-infected neurons was associated with polynucleated cells. Morphological and nu-
clear alterations on HTLV-1-infected cells have been previously reported. Indeed, Tax
was reported to induce genetic instability and increase the frequency of mutations,
thereby inducing important morphological and functional changes that resulted in
micro/multinuclei development (14–16). The impact of Tax-induced chromatin modifica-
tions on neuronal survival and function remains to be addressed. Cognitive dysfunction
FIG 2 Detection of the HTLV-1 viral proteins in the CNS of naturally SLTV-1-infected NHP. (A)
Sections from the spinal cord, cortex, and cerebellum of naturally STLV-1-infected NHP expressing the
Tax protein (arrowheads). Scale bar: 50
m
m. (B) Colocalization of the Tax and Gag p19 proteins of
STLV-1 in spinal cord sections of NHP. Scale bar: 20
m
m.
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in some HTLV-1-infected individuals (17) might be reminiscent to neuronal infection. Most
neural HTLV-1 manifestations are observed in HAM/TSP patients who are infected during
adulthood. However, the HTLV-1 neuroinfection of newborns during mother-to-child trans-
mission (18) could lead to more severe defects, as neural progenitors are more susceptible
to cell cycle deregulations or induced apoptosis. Several cases of HAM/TSP were reported
in adolescents who were breastfed by mothers infected with HTLV-1 (19), suggesting that
the impact of HTLV-1 infection on neural development may be underestimated.
The in vivo assessment of HTLV-1 neurotropism remains a challenging task due to the
complexity of accessing CNS samples from HTLV-1-infected individuals. The in situ
hybridization of spinal cord postmortem tissue from HAM/TSP patients revealed the
presence of HTLV-1 DNA in astrocytes from the spinal cord (10). However, the infection
of other resident cells of the CNS was not reported. The analysis of demyelinated sam-
ples may neglect the susceptibility of different cellular populations that are sensitive to
cell death or tissue atrophy. The analysis of postmortem CNS samples from naturally
infected NHP allowed us to detect the presence of the HTLV-1 Tax and Gag p19 proteins
in different neuronal subtypes in vivo, suggesting the productive infection of neurons.
While several neuronal subpopulations were found to be positive for STLV-1 infection,
such as pyramidal and motor neurons, granular neurons remained Tax negative. A differ-
ential tropism among neuronal populations was suggested to occur with other viruses,
such as Vesicular Stomatitis Virus (VSV), in which serotonin and norepinephrine neurons
were selectively infected (20). The mechanisms driving the selective STLV-1 neurotro-
pism remain unknown, but they could respond to (i) opportunity (access to a certain
FIG 3 Infection of neural cell populations in naturally STLV-1-infected NHP. (A) Expression of Tax
protein (arrowheads) in astrocytes (GFAP
1
, asterisks), microglial cells (Iba1
1
), and oligodendrocytes
(Olig2
1
, asterisks). Scale bar: 50
m
m. (B) Tax protein expression in NeuN
1
neurons from the spinal
cord, cortex, and cerebellum. Scale bar: 20
m
m. (C) Bar plot representing the frequency of Tax
1
cells
in the NeuN
1
, GFAP
1
, and Iba1
1
cells from spinal cord sections of NHP.
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region), (ii) the absence of restriction factors that allow for cell infection in susceptible neu-
rons, and/or (iii) the differential expression of viral receptors that allow for cell entry.
Neuropilin-1, one of the known viral receptors of HTLV-1, is highly expressed by endothe-
lial cells and neurons (21), and it could be utilized to facilitate HTLV-1 infection. Indeed, the
infection of endothelial cells seems to be a plausible route of entry into the CNS (22, 23).
The high frequency of infected cells in the spinal cord of asymptomatic NHP, together
with the infection of the cortical and cerebellar regions, suggests a more extensive viral
spread within the CNS than it was previously reported. Magnetic resonance imaging
(MRI) has revealed lesions in several CNS areas, such as cortical white matter regions,
even before symptomatic manifestations (24), suggesting that demyelination is not re-
stricted to the spinal cord. Activated microglial cells were found in close apposition of
Tax
1
cells in infected areas and may reflect the sensing and induction of an immune
response to the infection. The engulfment of infected neurons could trigger inflamma-
tion within the CNS, together with their clearance in later stages. Multiple and quite
diverse neuropathies, other than HAM/TSP typical symptomatology, have been reported
in HTLV-1 seropositive subjects, such as acute disseminated encephalomyelitis, meningi-
tis, myopathies, or peripheral neuropathies (25, 26), the development of which are still
not well-understood. These pieces of evidence lead us to hypothesize an important role
of microglia in neuroinflammation and interrogate whether its response to neuronal
infection could be at the origin of HTLV-1-associated neuropathies and at the onset of
HAM/TSP. In conclusion, our results showed that the impact of HTLV-1 neuroinfection
might be underestimated, as HAM/TSP manifestation is only the late stage of a progres-
sive, aggressive, and complex immunological response to neuroinfection. Furthermore,
our study emphasizes the use of naturally STLV-1-infected NHP as a relevant model with
which to investigate STLV-1 invading and spreading within the CNS. This approach
would pave the way for the identification of biomarkers of neuroinflammation before
HAM/TSP symptomatic manifestations.
MATERIALS AND METHODS
Ethics statement. The use of animals was approved by the Ethics Committee No. 14 (APAFIS no.
4227–201604130940121) of the French Minister of Education and Research. Animals were housed at the
primate center of the CNRS (UAR 846) in Rousset-sur-Arc and were cared for in compliance with French
regulations. The experimental procedure complied with the current French laws and the European direc-
tive 86/609/CEE. Blood was obtained after anesthesia via the intramuscular injection of ketamine (5 mg/kg)
and medetomidine (0.05 mg/kg), and CNS samples were obtained after euthanasia via the intravenous injec-
tion of pentobarbital (180 mg/kg).
FIG 4 Neuroimmune response to HTLV-1 infection. (A) Microglia cells (Iba1
1
) localized nearby Tax
1
cells in spinal cord sections of NHP. Arrowheads point to extended processes contacting infected
cells. Scale bar: 50
m
m. (B) Microglial cells (Iba1
1
) expressing the activation marker CD40 Scale bar:
50
m
m. A one-way ANOVA that was followed by a Fisher’s post-test was performed to determine
statistical significance. ****,P,0.0001.
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Animals. Eight naturally STLV-1-infected baboons (Papio anubis) were included in this study. Two
animals were STLV-1 naive. HTLV-1 serology was performed to discriminate HTLV-1-positive animals
from HTLV-1-negative animals. Then a polymerase chain reaction (PCR) was performed on the positive
animals to determine the proviral load (PVL). All of the analyzed animals were females, and the average
age at the moment of the euthanasia was 23 years.
Proviral load measurement. The PVL was measured from isolated PBMC. Briefly, PBMC were iso-
lated from total blood via Ficoll gradient after centrifugation. After two washes in phosphate-buffered
saline (PBS), the cells were centrifuged. Genomic DNA was extracted from the cell pellets using a
NucleoSpin Blood Kit (Macherey-Nagel, Düren, Germany). The DNA concentration was determined using
a NanoDrop ND-1000 spectrophotometer (Thermo Scientific).
Real-time PCR was performed with 20 to 40 ng of genomic DNA for controls as well as 100 to 120 ng
for virus amplification, using the FastStart Universal SYBR Green Master (Roche, reference number
4913850001), in 50
m
Lofthefinal volume. The primer sequences for the viral amplification were selected
in the tax gene: F-59-GTTGTATGAGTGATTGGCGGGGTAA and R-59-TGTTTGGAGACTGTGTACAAGGCG. The
primer sequences for control amplification were selected on the
b
-actin gene (27). The STLV-1 copy num-
bers were determined as the number of copies for 1 10
5
cells. The sensitivity was determined to be 10
copies of the Tax amplicon.
CNS sample processing and immunofluorescence. The brain and the first segment of the spinal
cord were harvested after the euthanasia of the animals. The spinal cord, motor cortex, and cerebellum
were dissected and fixed in 4% paraformaldehyde (PFA) for 48 h. The samples were embedded in paraf-
fin wax (Sigma-Aldrich, catalog number P3558) and sectioned at 5
m
m. The slides were dewaxed in
xylene (VWR; 3 times for 5 min) and rehydrated in successive baths of EtOH 95%, 70%, and 30%. After 5
min in water, the slides were incubated with a sodium citrate solution (10 mM sodium citrate, Sigma;
0.05% Tween 20, VWR; pH = 6) in a boiling water bath for 20 min for heat-induced epitope retrieval and
were washed in PBS. The samples were then incubated with blocking buffer (PBS, 3% BSA, 0.15% Triton
X-100) for 20 min. Then, the sections were stained in blocking buffer overnight at 4°C with mouse anti-
Tax (1:100, courtesy of Y. Tanaka) (28), mouse anti-Gag p19 (1:100, Zeptometrix), rabbit anti-GFAP (1:500,
Dako), guinea pig anti-NeuN (1:500, Sigma), goat anti-Iba1 (1:200, Wako), polyclonal goat anti-hOlig2
(1:100, catalog number AF2418, R&D Systems) or rabbit anti-CD40 (1:100, Ozyme).
After 3 washes in PBS for 5 min, the slides were incubated with anti-mouse Alexa Fluor 488, anti-
goat Alexa Fluor 555, anti-guinea pig Alexa Fluor 555, and anti-rabbit Alexa Fluor 647 conjugated anti-
bodies as well as with DAPI (Invitrogen) that was diluted in blocking buffer (1:750, Life Science
Technologies). Finally, the sections were mounted on glass slides with Fluoromount G (SouthernBiotech)
before observation via confocal microscopy (Zeiss LSM800) using 40and 63oil objectives.
For the quantifications, triplicate sections of each animal were analyzed. Between three to five images
were analyzed in which positive cells were identified. The number of positive cells was divided by the total
number of cells per field. A one-way ANOVA followed by a Fisher’s post-test was performed to determine
statistically significant differences in the numbers of Tax
1
cells between neural cell populations.
Human iPSC-derived neural polycultures. Human embryonic fibroblasts (HEF), kindly provided by
Odile Boespflug-Tanguy (AP-HP, Robert Debre Hospital, Department of Neuropediatrics and Metabolic
Diseases, National Reference Center for Leukodystrophies, Paris, France), were reprogrammed into
hiPSCs using a CytoTune Sendai Reprogramming Kit (Life Technologies), according to the manufacturer’s
instructions. Once isolated, the clones were amplified, fully characterized, registered in the Human
Pluripotent Stem Cell Registry (https://hpscreg.eu/) and declared through the Codecoh DC-2020-3895.
Dissociated hiPSC cells were plated at 1.5 10
5
cells in a poly-L-ornithine/laminin-coated well containing a
neural induction medium (NIM) that was composed of DMEM/F-12 complemented with 2 mM L-glutamine,
1,000 U/mL penicillin-streptomycin, 1% MEM nonessential amino acids solution, 1 mM 2-mercaptoethanol
and 1% N-2 supplement. The medium was changed every 2 days, and the cells were changed into neural
stem medium that was supplemented with 20 ng/mL of human recombinant basic fibroblast growth fac-
tor (hrFGF; 154 AA, Peprotech) and 20 ng/mL of murine recombinant epidermal growth factor (mrEGF;
Peprotech) after 7 days. After 14 days, the medium was supplemented with 0.5
m
M ATRA, 2% B-27 supple-
ment, and 100 ng/mL of human recombinant Sonic Hedgehog (hrSHH, StemCell Technologies). At day 28,
the NIM was complemented with 2% B-27 supplement, 100 ng/mL of hrSHH, and 10 ng/mL of hrFGF for
2 days. The cells were then used for viral testing after 30 days.
HTLV-1-infected cell lines and cocultures. Polycultures derived from hiPSCs were differentiated
and matured for 30 days to ensure the presence of neurons, astrocytes, and oligodendrocytes. The
obtained polycultures were cocultured with the HTLV-1-chronically infected lymphocyte cell line C91-PL
for 5 days at 2 different cell ratios (1:10 and 1:2). C91-PL cells were previously incubated with mitomycin
(50
m
g/mL, Sigma) for 20 min to stop the progression of the cell cycle. After 23 h, the cultures were
flushed with PBS, and the medium was changed every other day. On the fifth day, the cells were fixed
with 4% PFA and rinsed with PBS. Staining using anti-Tax (1:100) and anti-Tuj1 (1:500) antibodies was
performed directly in the ibidi 24-well plates (BioValley). Images were taken using an inverted micro-
scope Zeiss Axio Observer Z1 with a confocal unit LSM 980, and the images were analyzed using the
ImageJ software 1.52p Fiji package.
ACKNOWLEDGMENTS
The authors would like to dedicate this work to the memory of Renaud Mahieux
(1968 to 2020), who supported this work, helped with its design, and specially provided
access to the NHP cohort. This work was supported by Ligue Contre le Cancer (Equipe
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March/April 2023 Volume 14 Issue 2 10.1128/mbio.03526-22 7

labelisée program EL2013-3 Mahieux), Fondation pour la Recherche Médicale (FRM,
program Equipe labelisée, program DEQ20180339200), University of Lyon (Fapesp 2014/
22827-7 joint program 2015 and IDEX-INT-2020-36). B.R. was supported by FRM, N.A. was
supported by FINOVI (no. 13), H.D. is supported by the French National Institute of Health
and Medical Research (INSERM), C.M. was supported by a French ANR NITRODEP grant
(project ANR-13-PDOC-0010-01) (http://www.agence-nationale-recherche.fr), and R.P.
was supported by the French Agence Innovation Défense (DGA-AID) and INSERM. The
funders had no role in the study design, data collection, data analysis, decision to publish,
or preparation of the manuscript.
We declare no conflict of interest.
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