Supplementary information – Material and Methods (Material e-1):

JC virus DNA quantification

JC virus DNA was analyzed from various biospecimens using established JC-virus

specific primers (1). Freshly frozen autopsy specimens were taken from several brain

regions, from sternal bone marrow and from kidney. After digestion with proteinase K

(Tissue Kit, Qiagen), DNA was separated from the centrifuged cell-free supernatant

(EZ-1-Biorobot, Qiagen) and processed by PCR with JC-virus specific primers. The

complete procedure was performed in a certified laboratory for clinical diagnostics

(University of Düsseldorf DAR registration number DAC-ML-0502-07-00).

Immune cell purification and -isolation

Peripheral blood mononuclear cells (PBMC) were isolated via density gradient

centrifugation using lymphocyte separation medium (PAA Laboratories, Linz, Austria)

within 24 hours after blood draw. CD4+ or CD8+ T cells were negatively isolated

from PBMC using magnetic bead isolation (negative isolation procedure, MACS®,

Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer‘s

instructions. Purified T cell subsets were over 95% pure as controlled by flow

cytometry analysis.

Culture of effector cells grown from PBMC

PBMC of patient 1 obtained before PE and one week after the completion of PE were

stimulated with HLA-A*0201/JCV VP1p36 peptide (2) at 1 mg/ml and cultured for 14

days in RPMI supplemented with 8% human serum in the presence or absence of 20

U/ml of recombinant human IL-2.

Tetramer staining

After culture, CD8+ T cells were tested for JCV specificity by staining them at 4°C for

30 minutes with the HLA-A*0201/JCV VP1p36 tetramer [obtained from the NIH

Tetramer Core Facility at Emory University (Atlanta, GA)]. Anti-CD3-PerCP-Cy5.5

and anti-CD8-APC (both from Becton Dickinson) were then added for 20 minutes at

4°C. Cells were washed and data were acquired on a LSRII flow cytometer (Becton

Dickinson, Franklin Lakes, NJ) and analyzed using FlowJo Software (Tree Star Inc.,

Ashland, OR).

Intracellular Cytokine Staining assay

PBMC effector cells were cultured for 14 days. Cells were washed and rested

overnight in RPMI supplemented with 10% FCS. Cells were then restimulated with

the HLA-A*0201/JCV VP1p36 peptide for six hours. An ICS assay was performed

such as previously described (3) with the following antibodies: CD3-APC-Cy7, CD8-

Pacific blue, IFN-g-APC, IL-2-PE (depending on the figure), TNF-a-FITC (all from

Becton Dickinson) and CD4-ECD (Beckman Coulter). Dead cells were excluded

using the Aqua LIVE/DEAD stain kit (Invitrogen, Basel, Switzerland). Data were

acquired on a LSRII flow cytometer (Becton Dickinson) and analyzed using FlowJo

Software (Tree Star Inc.).

Preparation of cells from CSF samples

CSF (8-10 ml) was obtained by lumbar puncture and processed as previously

described (4,5). In brief, after collection of CSF, cells were obtained by centrifugation

at 200 g for 20 min at 4oC. The supernatant was removed, and the CSF cells were

washed once in RPMI containing 10% FCS; Isolated PBMC were used as a control.

Reagents and flow cytometry

The following mAbs were used: CD4-FITC/-PE/-PerCP, CCR5-PE-Cy5/-APC, CCR7-

FITC/-APC, CD27-FITC, CD45RA-APC, CD45RO-FITC (all BD Pharmingen

Bioscience Heidelberg, Germany). For flow cytometry, cells were resuspended in ice-

cold PBS containing 1% bovine serum albumin (BSA) and 0.5% sodium azide and

stained with fluorescence-labeled mAbs at 4oC for 30 minutes. Cells then were

analyzed with FACS-Calibur™ using Cell Quest™ (Becton Dickinson, Heidelberg,

Germany) and FlowJo (Tristar, USA) software. For intracellular staining, the cells

were fixed and permeabilized with PFA/Saponin (BD Biosciences).

Human brain microvascular endothelial cell (HBMEC) cultures and transmigration

assay

HBMEC were purchased from ScienceCell Research Laboratories (San Diego, CA).

Cells were cultured on filter membrane of Transwells® (3-µm pore-size, (Corning, NY,

USA) until reaching confluence. Transmigration assays were performed as described

previously (6,7). Briefly, HBMEC were cultured on the apical side of a filter

membrane of Transwells® till confluence. HBMEC were pre-incubated with TNF-alpha

and IFN-gamma (500U/ml for each, 6 hours, 37°C) prior to the experiments. 2.5 x 105

of purified T cells in 0.1 ml of pre-warmed RPMI medium were added to the top of the

HBMEC monolayers and 0.6 mL of media was added in the outer chamber of the

inserts. The cells were allowed to migrate for 18 h in a humidified cell culture

incubator at 37oC and 5% CO2.

RNA isolation and cDNA synthesis:

RNA from blood and CSF immune cells was isolated using standard methods

(Quiagen RNeasy kit; Quiagen, Hilden, Germany) according to the manufacturer‘s

instructions. Isolation of RNA from tissue specimens was performed from cryo-

preserved CNS tissue. Slices were directly transferred into TriZol (Invitrogen,

Mannheim, Germany), homogenized, and then processed according to the

manufacturer‘s protocol.

cDNA synthesis from peripheral blood T cell RNA was performed using standard

methods as previously described (8). Reagents were obtained from Applied

Biosystems (Foster City, CA, USA). For each sample 250ng of RNA were

transcribed, using random hexamers and M-MLV reverse transcriptase.

For RNA isolated from CNS samples, a previously published sensitive TCR β-chain-

specific cDNA primer ‚Cβ-RT‘ (9) was used, as well as SuperScriptIII™ reverse

transcriptase (Invitrogen, Karlsruhe, Germany), according to the manufacturer‘s

instructions.

CDR3 spectratyping:

The PCRs have been performed as described previously (10). In brief, for the CDR3-

spectratyping, we used the Vβ forward primers as described in Monteiro et al. (11),

the Jβ reverse primers as described previously (12), and two different Cβ reverse

primers: ‚SpTy-β-out‘ (13) and ‚Cβ-R‘ (11). The Vβ nomenclature according to Arden

et al. (14) is used throughout the manuscript.

For the peripheral blood derived samples, the following protocol was applied: First,

the cDNA was used in 25 Vβ-Cβ reactions: 1.25µl 10pmol/µl Vβ primer, 1.25µl

10pmol/µl Cβ-R primer, 0.5µl cDNA, 0.25µl 2.5mM dNTPs (Promega, Mannheim,

Germany), 2.5µl 10xbuffer, 0.1µl TaqPolymerase (both Applied Biosystems), 19.15µl

DEPC-H2O. PCR-conditions: 94°C, 6min; 94°C, 1min, 59°C, 1min, 72°C, 1min (40x);

72°C, 7min. After those first-round PCRs, every PCR product was subjected to 13

individual Vβ-Jβ ‚run-off‘ reactions (modified from (15,16)) with 13 5‘-fluorescence-

tagged Jβ primers to differentiate between individual TCR Jβ-regions and also with a

5‘-fluorescence-tagged Cβ-R primer. The length of these fluorescence-labeled PCR

products was then analyzed on an ABI3130 genetic analyzer (Applied Biosystems),

applying a module for fragment analysis. 500-ROX (Applied Biosystems) was the

internal standard in each sample.

In the case of the CNS-samples, we used a more sensitive protocol (13) to

compensate for the low T cell numbers in the CNS specimens. Briefly, we introduced

a semi-nested pre-amplification PCR step before the PCR reaction described above.

There, we used the same forward primers as above, but employed the Cβ-specific

reverse primer ‚SpTy-β-out‘ (10pmol/µl) that hybridizes downstream of CβR, but

upstream of the RT-primer Cβ-RT. Samples were incubated for 5min at 94°C. Then

PCR was run for 30 cycles of: 94°C, 1min, 56°C, 1min, 72°C, 1min, followed by an

incubation of 72°C, 10min. From this PCR, 1µl/reaction was used as template in the

protocol mentioned before. NED-tagged primers were bought from Applied

Biosystems (Foster City, CA, USA) and all other primers were provided by Metabion

(Martinsried, Germany).

To briefly summarize our method of analysis, we compared the number of normally-

distributed TCR families (Normal/Gaussian distribution [GD] of TCR CDR3: 8 or more

peaks in the CDR3 histogram were considered to be GD) to the number of expanded

TCRs (3 or less peaks in the CDR3 histogram were considered a clonal expansion).

Histograms that could not be clearly assigned to either group were omitted from the

analysis to simplify the manuscript.

Examples for CDR3 histograms.

Neuropathological workup and JC detection in tissue specimens:

Brains from two deceased patients treated with efalizumab were removed and

partially dissected in the fresh state to flash freeze selected portions, and the rest

was fixed in 10% neutral buffered formalin. After fixation and brain cutting, tissue

regions were processed into paraffin blocks, and selected sections were stained with

hematoxylin and eosin (HE), luxol fast blue-HE (LFB-HE), Bielschowsky silver stain,

and immunohistochemistry for myelin, axons, inflammatory cell, and JC virus

antigens (see Table e-2). Digital images were processed using Photoshop (Adobe,

San Jose, CA).

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4. Huang Y, Zozulya AL, Weidenfeller C, Metz I, Buck D, Toyka KV, et al. Specific central nervous system recruitment of HLA-G(+) regulatory T cells in multiple sclerosis. Ann Neurol. 2009 Aug. 1;66(2):171-183.

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6. Debes GF, Arnold CN, Young AJ, Krautwald S, Lipp M, Hay JB, et al. Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues. Nat Immunol. 2005 Sep.;6(9):889-894.

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9. Seitz S, Schneider CK, Malotka J, Nong X, Engel AG, Wekerle H, et al. Reconstitution of paired T cell receptor alpha- and beta-chains from microdissected single cells of human inflammatory tissues. Proc Natl Acad Sci USA. 2006 Aug. 8;103(32):12057-12062.

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12. Puisieux I, Even J, Pannetier C, Jotereau F, Favrot M, Kourilsky P. Oligoclonality of tumor-infiltrating lymphocytes from human melanomas. J Immunol. 1994 Sep. 15;153(6):2807-2818.

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16. Pannetier C, Cochet M, Darche S, Casrouge A, Zöller M, Kourilsky P. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments. Proc Natl Acad Sci USA. 1993 May 1;90(9):4319-4323.

Table e-1. Immunological workup of case 1

Given are percentages of immune cells characterized as either CD4+, CD8+,

TEM/CD45RA-CCR7- (shaded grey), CD34+ or regulatory T cells (Tregs) and

belonging to either the periphery, the CSF (values in parentheses) ex vivo, the

upper or lower compartment of a migration assay and compared to HIV+

cases with PML or a mean of 3 healthy donors (HD).

Table e-2: Antibodies and staining conditions for histology

Antibodies/target antigen (Laboratory)

source dilution/ pretreatment

Macrophage markers

mouse anti-CD68 Dako 1: 50/protease

mouse anti-KiM1P Prof. Radzun, Department of Pathology, University of Göttingen

1: 5000/microwave

mouse anti-CD68 (CCF) DAKO, clone PG-M1, M0876 1:100/ microwave, citrate buffer pH 6.0

T cell markers

Rat anti-CD3 Serotec 1:100/microwave

mouse anti-CD8 DCS Undiluted/EDTA pH 8

mouse anti-CD4 DCS Undiluted/EDTA pH 8

rabbit anti-CD3 (CCF) Novocastra, NCL-CD3p 1:100/microwave, citrate buffer pH 6.0

mouse anti-CD8 (CCF) Vector Laboratories, VP-C325 1:100/ microwave, citrate buffer pH 6.0

B cell markers

mouse anti-CD20 Dako 1: 50/none

mouse anti-CD79a Dako 1 : 50/ microwave

rabbit anti-CD20 (CCF) AbCam, ab27093 1:100/microwave, citrate buffer pH 6.0

Plasma cell marker

mouse anti-CD138 Dako 1: 100/microwave

mouse anti-CD138 (CCF) Biocare Medical, CM167C 1:200

Astrocyte marker

mouse anti-GFAP Dako 1 : 50/none

Antibodies against JV virus

rabbit anti-JC virus (PAB2003)

Prof Frisque, Department of Biochemistry and Molecular Biology, Pennsylvania State University

1: 300/none

Myelin markers

rabbit anti-MBP Dako 1 : 1500/none

mouse anti-CNPase Sternberger Monoclonals 1 : 200/microwave

mouse anti-MBP (CCF) Covance, SMI-94 1:100/microwave Citrate Buffer pH6.0

Figure e-1 : MRI scans of the efalizumab-associated PML case 2

Figure e-2: Clonal distributions in patient 1.

A: Shown are T cell expansions that exist in more than one compartment.

CNS expansions are shown in black, CSF expansions in green, peripheral

CD4+ T cell expansions in blue, and peripheral CD8+ T cell expansions in

red. Identical clones have been checked by DNA sequencing.

B: Clonal expansions in the previously described autopsy samples

(schematics see figure legend figure 3) are listed. The upper line lists the

analyzed Vβ families, the left column depicts the specific CNS sample (by

number and overriding region). Squares indicate a dominant clonal

expansion, triangles show oligoclonal expansions where another expansion

appeared in the same Vβ. GD shows indicates normal Gaussian distribution.

Green symbols indicate a dominant clonal expansion also found in the same

Vβ of the CSF compartment. Red symbols are expansions with corresponding

expansions in peripheral blood CD8+ T-cell spectratyping, yellow symbols

stand for corresponding expansions in the peripheral blood CD4+ T-cell

repertoire.

Figure e-3: Hypothetical sequence of events – Effects of efalizumab on

the control of JC virus infection in periphery and CNS

Active replication of JC virus usually initiates antiviral immune responses.

Whether this is the reactivation of latent virus or a novel infection (e.g. by

other archetypes) is not known. In either case, the host is confronted with

virus-infected cells in the periphery. Due to virus replication eventually virus

antigens are presented to T cells. This occurs e.g. after release of viral

particles into the system, where they are phagocytosed and presented by

immature dendritic cells (DC). After migration of these DC to peripheral lymph

nodes, priming of naive JC specific T cells (CD4 and CD8) occurs, their

activation into effector phenotypes requires recognition of the cognate

antigens (e.g. in tissues infected by the virus). The JC specific CD8 effector

cells are then able to kill infected cells efficiently.

JC virus enters the CNS via migration of infected cells or by the invasion of

soluble virus particles. However, to eliminate or control JC in the CNS,

effector T cells have to be generated and to migrate via the blood-CSF or

CSF-brain barrier. Once virus particles are in the CNS, oligodendrocytes are

susceptible to JC infection. They can be damaged by autolysis, which could

lead to the release of non JC related epitopes in the CNS and to

demyelination, or they can be killed by JC specific CD8 T cells, which leads -

in the best case - to virus clearance, but can also enhance virus load due to

the release of particles from the infected cells.

Expanded T cells also migrate from the periphery to the CNS over the Blood-

CSF barrier. In the CSF they make contact with meningeal macrophages,

which upon encounter of virus antigens via the draining pathways of the CNS

secrete cytokines to activate the BBB and these macrophages are also

imperative to restimulate JC specific T cells leading to T cell expansions in the

CSF. We assume that efalizumab, a blocker of LFA-1 interactions, mainly acts

on 3 levels. 1) The priming of T cells in the lymph nodes is critically dependent

on the establishment of the immune synapse between T cells and DC (or T

cells and macrophages), which is why efalizumab can possibly prevent T cell

activation and the shift into the effector phenotype. 2) Also, CD8 mediated

killing of infected cells is inhibited by anti-LFA-1. 3) Most importantly, the

migration of T cells over the BBB is abrogated by the blockade of LFA-1,

which inhibits the primary wave of T cells.