CD4+ T cells in immunopathogenesis of PML-IRIS
Supplementary material for:
Central Role of JC Virus-Specific CD4+ Lymphocytes in PML-Immune
Reconstitution Inflammatory Syndrome
Lilian Aly*1, Sara Yousef*1, Sven Schippling1, Ilijas Jelcic1,2, Petra Breiden1, Jakob
Matschke3, Robert Schulz4, Silvia Bofill-Mas5, Louise Jones6, Viktorya Demina7,
Michael Linnebank2, Graham Ogg6, Rosina Girones4, Thomas Weber8, Mireia
Sospedra**1, Roland Martin**1
Supplementary Material and Methods
Other PML-IRIS-, PML patients and inflammatory neurological controls
PML- and PML-IRIS patients not related to multiple sclerosis and natalizumab
treatment
PML and PML-IRIS in a case of AIDS – AIDS was diagnosed in this 48 year
old male patient 3/2010. At this time-point MRI showed a large T2-hyperintensive
lesion in the right precentral region and additional lesions in the frontal superior lobe
and precuneus, which were suggestive of PML, but JCV DNA in the CSF was
negative. 4/2010 he experienced a seizure, MRI showed progression, and a
diagnosis of PML was made based on positive CSF JCV DNA. Despite antiretroviral
treatment the patient deteriorated during the next two months with progressive
hemiparesis and mental confusion. 6/2010 MRI showed multiple T2-hyperintensive
lesions in the left hemisphere and ventral pons with extensive and space-occupying
edema including diffuse contrast enhancement. Although PML-IRIS was the most
likely cause of these changes, CNS lymphoma could not be ruled out, and therefore
a brain biopsy was performed. Histopathology showed SV40-positive inclusion
bodies within oligodendrocytes, positive PCR for JCV DNA and inflammatory
infiltrates (CD4+ and CD8+ T cells, and CD20+ B cells) without detection of “cell
blasts”. Consequently, PML-IRIS was diagnosed. The patient died of uncal
herniation due to mass effect of the PML-IRIS-associated edema in 7/2010.
PML in a case of idiopathic CD4 lymphopenia - The previously healthy 63
year old male developed a subacute cerebellar and brainstem syndrome in 2/2010.
Cerebral MRI showed marked T2 hyperintensities bilaterally in the cerebellum. CSF
analysis revealed identical bands in serum and CSF, but was otherwise
unremarkable. CSF-PCR for neurotropic viruses was negative. The patient did not
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CD4+ T cells in immunopathogenesis of PML-IRIS
respond to a three days course of methylprednisolone (1g/day). To rule out a
neoplastic disease, the patient underwent a cerebellar brain biopsy that showed no
signs of malignancy. Based on positive EBV-PCR from brain tissue (300 copies) a
diagnosis of EBV cerebellitis and brainstem encephalitis was made and Ganciclovir
therapy initiated. Following transient slight improvement the patient deteriorated
further, and MRI (9/2010) showed new T2 lesions bilaterally in the temporal and also
right parietal subcortical white matter. Isolated CSF oligoclonal bands and slightly
elevated protein (566 mg/l) were now detectable. Due to diagnostic uncertainties
and radiological deterioration despite sufficient Ganciclovir treatment, a second brain
biopsy from a left frontotemporal lesion was performed. Immunohistochemistry
showed SV40- and Pab2003-positive nuclei confirming PML. Immunological work up
showed low (300/µl) absolute CD4+ T cell counts and a CD4/CD8 ratio of 0.5 despite
negative HIV serology. Based on these findings a diagnosis of PML on the basis of
idiopathic CD4 lymphopenia was made. Until 2/2011 the patient progressed clinically
and developed aphasia, which had begun after the second brain biopsy.
PML in a case of hyper-IgE syndrome - The 20 year old male patient with
known hyper-IgE syndrome (STAT3 mutation analysis negative) developed left sided
sensory deficits in 8/2008. The MRI showed a confluent T2- and FLAIR
hyperintensity in the right postcentral region extending into the medulla oblongata.
CD4+ T cells (71/µl) and absolute lymphocyte counts (296/µl) were markedly
decreased with at the same time massively elevated eosinophils (6068/µl). JC virus
PCR proved repeatedly positive in the CSF (800 copies/ml). The patient was
diagnosed with PML and received 11 cycles of Cidofovir between 11/2008 and
4/2009. Clinically, he developed a slowly progressive left-sided weakness and
symptomatic focal epilepsy. He was treated with interferon- 1b June and July 2009.
CSF JCV viral load increased until 9/2009 (5.200 copies/ml), and the patient
worsened significantly with progressive hemiparesis and dysarthria and an
increasing PML lesion in the right temporal and parietal lobe. He was admitted to a
nursing home 12/2010.
Non-PML Patients –Controls:
Patient with optic neuritis and neurolues - The 50 year old male presented
with left-sided blurred vision and central scotoma 10/2010. His prior medical history
was unremarkable except for arterial hypertension. CSF analysis showed
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CD4+ T cells in immunopathogenesis of PML-IRIS
pleocytosis with 280 cells/µl, elevated protein (538 mg/l) and oligoclonal bands.
Serology revealed positive TPHA (serum 1:80.000; CSF 1:4.096) and VDRL (serum
1:32; CSF 1:8) tests with a positive IgG-index (4.240) confirming a diagnosis of
neurolues. The patient received i.v. antibiotic treatment. Follow-up CSF analysis
showed a decreased cell count (45 cells/µl), and visual acuity improved under
concomitant steroid treatment.
Patient with relapsing-remitting multiple sclerosis - The 32 year old female
developed paraparesis in 2010. A cranial MRI revealed 25 hyperintense T2 lesions.
Oligoclonal bands were exclusively positive in CSF, and the spinal MRI also showed
non-enhancing T2 hyperintensities. A diagnosis of relapsing-remitting MS (RRMS)
was confirmed by proving dissemination in time in a follow-up cranial MRI 2/2011.
The patient refused to initiate immunomodulatory treatment at that stage.
Quantification of JCV loads
Diagnostic testing for JCV viral loads in urine, serum and CSF samples of
patient 1 and the PML/PML-IRIS patient with AIDS was performed in the laboratory
of Prof. H.H. Hirsch (Transplantation Virology, Institute for Medical Microbiology,
Department of Biomedicine, University of Basel, Switzerland). JCV loads were
quantified by PCR-based molecular assays as previously described (Dumoulin and
Hirsch, 2011).
For patient 2 and the idiopathic CD4+ lymphopenia patient JCV viral load
testing was kindly performed by Dr. E.O. Major (Laboratory of Molecular Medicine
and Neuroscience (LMMN), NINDS, NIH Bethesda, USA). LMMN established and
maintains Clinical Laboratory Improvement Amendment (CLIA)-validated and
certified real-time quantitative polymerase chain reaction (qPCR) assays for the
detection of the JCV genome in clinical samples (Linda et al., 2009). The assay
targets a highly-conserved region of the viral genome that codes for the aminoterminal region of the multifunctional T protein, which is required for successful viral
infection. The limit of detection of the assay is 10 copies of the viral genome per
milliliter. The real-time PCR protocols and primers for detection of JCV DNA have
been previously described (Ryschkewitsch et al., 2004).
Supplementary Figure 1:
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CD4+ T cells in immunopathogenesis of PML-IRIS
MRI imaging of PML-IRIS patient 1: Axial MRI from 2009, a) 07/22 at time of
diagnosis of PML, showing a confluent occipital T2-weighted hyperintense lesion in
the left subcortical paramedian occipital area with minimal contrast enhancement, b)
09/15 at the time of diagnosis of PML-IRIS showing a broad progressive edema
around and an irregular contrast enhancement in the left parietooccipital lesion, the
right lobus parietalis superior, the right gyrus temporo-occipitalis and the white
matter of the right cerebellar hemisphere, c) 09/24 after beginning of corticosteroid
treatment on 09/17 MRI showed less contrast enhancement and decreasing edema.
Supplementary Figure 2:
MRI imaging of PML-IRIS patient 2: Axial MRI from 1/2010 (a), at time of
suspected PML with minimal contrast enhancement (middle panel), 2/2010 (b), and
at the time of biopsy (c) 3/2010. A large confluent, T2-hyperintense lesion is
observed that extends from the right parieto-occipital cortex forward into frontoparietal areas. Patchy contrast enhancement is observed early on, which later
expands significantly. At the time of biopsy (c), the large T2-hyperintense lesion
impresses also by T1 hypointensity and midline displacement as an indicator of
brain swelling.
Supplementary Figure 3:
Clinical course of PML-IRIS patient 2. 1Results provided by Prof. H. Hirsch,
University of Basel. 2Results provided by Dr. E.O. Major, NINDS, NIH, Bethesda.
Supplementary Figure 4:
a) Schematic depiction of PHA expansion of brain biopsy-derived material. b)
CD4+/CD8+ ratio of PHA-expanded brain biopsy-derived T cells after repetitive
rounds of stimulation. c) TCR V chain expression of CD4+ and CD8+ T cell
populations after various rounds of stimulation.
Supplementary Figure 5:
a) Proliferative response of PHA-expanded bulk mononuclear cell populations
from brain biopsy against 82 peptide pools with a two-dimensional pool setup and
seeding scheme, in which each individual peptide appears in exactly two pools and
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CD4+ T cells in immunopathogenesis of PML-IRIS
the remaining peptides are different. Results show the mean SI  SEM. b) The
analysis of positive pools allows the identification of immunogenic candidate
peptides at the intersections of the positive pools. c) Proliferative response of PHAexpanded bulk mononuclear cell populations from brain biopsy against individual
peptides and identification of actual stimulatory peptides. Results show the mean SI
 SEM.
Supplementary Figure 6:
a) Proliferative response of PHA-expanded bulk mononuclear cell populations
from CSF (upper panel) and PBMC (lower panel) tested against 204 overlapping 15mer peptides spanning all open reading frames of JCV (covering Agno, VP1, VP2,
VP3, Large-T, and small-T proteins) and organized in 41 pools of 5 peptides each.
Results show the mean SI  SEM. b) Reactivity of PHA-expanded CSF- and PBMCderived cells against JCV peptides that were recognized by brain-infiltrating T cells.
Results show the mean SI  SEM. Only peptide 191 (LTAg668) was recognized
above threshold levels by CSF-derived cells.
Supplementary Figure 7:
Proliferative response of PHA-expanded bulk mononuclear cell populations
from brain biopsy against sets of overlapping peptides covering the myelin proteins
myelin basic protein (MBP) (top panel), myelin oligodendrocyte glycoprotein (MOG)
(middle panel), and proteolipid protein (PLP) (bottom panel), as well as VP1/VLP,
tetanus toxin (TTx) and PHA. Results show the mean SI  SEM. None of the myelin
peptides is recognized.
Supplementary Figure 8:
Th1-2 and Th1 T cytokine secretion patterns are comparable after VP1/VLPand PHA stimulation. a) Intracellular cytokine staining for IFN- and IL-4 in one Th12 TCC (upper left panels) and one Th1 clone (lower left panels) respectively 12 days
after stimulation with VP1/VLP (left dot plots) and PHA (right dot plots). b) ELISA
detection of IFN- and IL-4 production in culture supernatants of two representative
Th1-2 TCC (21a and 30a) and two Th1 (16a and 7b) 72 h after stimulation with
VP1/VLP and PHA.
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CD4+ T cells in immunopathogenesis of PML-IRIS
Supplementary Table 1:
Patient 1: Summary of clinical-, neuroimaging- (MRI), virological, and clinical
chemistry findings in patient 1 with PML-IRIS, a 41year-old, Caucasian male.
Supplementary Table 2:
Patient 2: Summary of clinical-, neuroimaging- (MRI), virological, and clinical
chemistry findings in patient 2 with PML-IRIS, a 43 year-old, Caucasian male.
Supplementary Table 3:
Set of 204 overlapping 15-mer peptides covering all open reading frames of
JCV.
Supplementary Table 4:
82 pools of 204 15-mer peptides covering all open reading frames of JCV.
Pools are built in a way that each JCV peptide appears in two different pools of
peptides, however, the other four peptides in the two pools are different. Using this
setup- and seeding scheme, T cell responses to individual peptides can immediately
be identified (for details see methods).
Supplementary Table 5:
3 sets of overlapping peptides covering the three myelin proteins MBP, MOG and
PLP.
Supplementary references:
Dumoulin A, Hirsch HH. Reevaluating and optimizing polyomavirus BK and JC
real-time PCR assays to detect rare sequence polymorphisms. J Clin
Microbiol 2011; 49: 1382-8.
Linda H, von Heijne A, Major EO, Ryschkewitsch C, Berg J, Olsson T, et al.
Progressive multifocal leukoencephalopathy after natalizumab
monotherapy. N Engl J Med 2009; 361: 1081-7.
Ryschkewitsch C, Jensen P, Hou J, Fahle G, Fischer S, Major EO. Comparison of
PCR-southern hybridization and quantitative real-time PCR for the
detection of JC and BK viral nucleotide sequences in urine and
cerebrospinal fluid. J Virol Methods 2004; 121: 217-21.
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