immunology.sciencemag.org/cgi/content/full/5/53/eabc7191/DC1

Supplementary Materials for

Gut microbiota–specific IgA+ B cells traffic to the CNS in active multiple sclerosis

Anne-Katrin Pröbstel*, Xiaoyuan Zhou, Ryan Baumann, Sven Wischnewski, Michael Kutza, Olga L. Rojas, Katrin Sellrie, Antje Bischof, Kicheol Kim, Akshaya Ramesh, Ravi Dandekar, Ariele L. Greenfield, Ryan D. Schubert, Jordan E. Bisanz, Stephanie Vistnes, Khashayar Khaleghi, James Landefeld, Gina Kirkish, Friederike Liesche-Starnecker, Valeria Ramaglia,

Sneha Singh, Edwina B. Tran, Patrick Barba, Kelsey Zorn, Johanna Oechtering, Karin Forsberg, Lawrence R. Shiow, Roland G. Henry, Jennifer Graves, Bruce A. C. Cree, Stephen L. Hauser, Jens Kuhle, Jeffrey M. Gelfand, Peter M. Andersen,

Jürgen Schlegel, Peter J. Turnbaugh, Peter H. Seeberger, Jennifer L. Gommerman, Michael R. Wilson, Lucas Schirmer, Sergio E. Baranzini*

*Corresponding author. Email: anne-katrin.proebstel@usb.ch (A.-K.P.); sergio.baranzini@ucsf.edu (S.E.B.)

Published 20 November 2020, Sci. Immunol. 5, eabc7191 (2020)

DOI: 10.1126/sciimmunol.abc7191

The PDF file includes:

Fig. S1. Gut microbial structure in MS and healthy controls (HC). Fig. S2. CSF IgA and CNS-infiltrating IgA+ B cells are enriched in active MS but absent in noninflammatory disease and healthy controls. Fig. S3. IgA-producing cells traffic across the BBB in active MS. Fig. S4. Assessment of gut mucosal homing markers and antimicrobial reactivity of CNS IgA-producing cells. Fig. S5. Assessment of reactivity against human brain tissue. Fig. S6. Epitope mapping mAb-1. Fig. S7. Commensal-specific IgA antibody-secreting cells (ASC) detected in human blood of patients with MS.

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/53/eabc7191/DC1)

Table S1. Clinical characteristics of patients and controls in the study (Excel spreadsheet). Table S2. IgA-SEQ data analysis (Excel spreadsheet). Table S3. Cluster, read count, and mutation analysis for B cell repertoire data (Excel spreadsheet). Table S4. List of glycan array epitopes (Excel spreadsheet). Table S5. Raw data spreadsheet (Excel spreadsheet).

Supplementary Materials:

Fig. S1. Gut microbial structure in MS and healthy controls (HC). A, B, Microbiome alpha-

diversity (Shannon index) and principal coordinate analysis (PCoA) plot of beta diversity

(weighted UniFrac). MS: inactive: n=25, active: n=11; HC: n=31. PERMANOVA. C, Volcano

plots of the significantly differential microbial operational taxonomic units (OTUs) between

IgA+ and IgA- in healthy controls. D, Barplot of PICRUSt predicted microbiome KEGG

orthologies identified significantly differential pathways between MS and HC specific to IgA+ or

IgA-, respectively (DESeq2, Benjamini-Hochberg correction for multiple comparisons, P ≤0.05).

Notably, the MS-associated IgA+ fraction showed enrichment for regulatory elements and

metabolic enzymes.

Fig. S2. CSF IgA and CNS-infiltrating IgA+ B cells are enriched in active MS but absent in

noninflammatory disease and healthy controls. A, Association of IgA CSF levels with active

brain lesions as measured by average gadolinium enhancing lesion volume on MRI. IgA CSF

levels increased with increasing average volume of gadolinium enhancing brain lesions (IgA

CSF: 102.7 μg/ml, 95% CI [8.5; 196.9 μg/ml]). CSF=cerebrospinal fluid, IgA=immunoglobulin

A, MRI=magnetic resonance imaging. B, A trend was observed between presence of

perivascular IGHA-expressing B cells and increasing number of CD68+ macrophages in active

MS lesions (left) as compared to chronic-inactive MS lesions (right). C, Absence of both IGHA-

and IGHG-expressing cells in meningeal and perivascular/parenchymal subcortical tissues of

neurodegenerative disease (MND, amyotrophic lateral sclerosis) and healthy control brains.

Fig. S3. IgA-producing cells traffic across the BBB in active MS. B cell receptor repertoire

analysis from sorted B cell subsets from peripheral blood (PB, red) and cerebrospinal fluid (CSF,

blue) from an active MS patient (bBCR1; n=1). DN=double negative, N=naïve,

(U)SM=(un)switched memory, PC=plasma cells. Edges (lines): clonal connections between B

cell subsets. PB-CSF connections: blue=clonal connections between IgA subsets, red=all other

clonal connections. Node size and edge thickness are relative to the number of Ig-VH clusters

and connections, respectively. Highlighted in gray are the PB-CSF plasma cell connections.

Note, trafficking of IgA-producing cells across the blood-brain barrier during active

neuroinflammation.

Fig. S4. Assessment of gut mucosal homing markers and antimicrobial reactivity of CNS

IgA-producing cells. A, CCR10 and ITGB7 expression in IGHA+ cells in healthy colon tissue as

compared to inflamed MS lesions; note IGHA+CCR10+ITGB7+ cells marked by yellow

arrowheads. B, BUGFlow. Individual CSF reactivities against autologous gut microbiota in

active and inactive MS patients (left). Gating strategy for CSF (right). C, CSF IgA microbiota-

reactivity in other diseases. MND=motor neuron disease, HC=healthy controls; One-way

ANOVA with Tukey’s multiple comparison test. D, Anti-E. coli antibody reactivity on

autologous gut bacteria (positive control). E, BUGFlow of CSF mAb-reactivity against bacterial

strains. F, Phylogenetic tree of enriched OTUs bound in autologous (left) and non-autologous

MS gut microbiota (right) by five mAbs (enrichment score > 0.5, relative abundance > 0.05%).

G, ELISA of CSF mAb reactivity against autologous intact and lysed bacterial antigens. H,

ELISA of CSF mAb-reactivity against self- and foreign antigens (hMOG=human MOG,

dsDNA=double-stranded DNA) and cell-based assay (CBA) with human MOG (mAb [0.5

μg/ml]).

Fig. S5. Assessment of reactivity against human brain tissue. Human anti-myelin

oligodendrocyte glycoprotein (MOG, clone 8-18-C5) IgG antibody staining (positive control)

shows specific myelin fiber staining in subcortical white and cortical grey matter areas (first

row). In comparison, recombinant anti-E. coli IgG (second row) and other recombinant human

(patient scBCR) IgG antibody stainings show no specific tissue reactivity (rows 3-5;

representative stainings from mAb-2, -4 and -5; mAb-1 and -3 not shown). For all stainings,

representative images from different tissue sections shown (n = 4; human control brain tissue).

Fig. S6. Epitope mapping mAb-1. A, Microbial glycan array printing outline and binding

profile with mAb-1. B, Epitope fine-mapping of mAb-1. (1) ManNAc(b1-3)FucNAc(a1-

3)GalNAc(a1-4)Gal(a1-1)aminopentanol - Depyruvated S. pneumoniae Serotype 4 repeating

unit; (2) ManNAc(b1-3)FucNAc(a1-3)GalNAc(a1-4)Gal-2,3-pyruvate(a1-1)aminopentanol -

Pyruvated SP4 repeating unit; (3) ManNAc(b1-4)Glc(b1-4)Glc(a1-1)aminopentanol - S.

pneumoniae ST9A CPS trisaccharide; (4) negative control: Printing buffer, NaP [pH 8.5]

[printing concentration (1-4): 100 µM].

Fig. S7. Commensal-specific IgA antibody-secreting cells (ASC) detected in human blood of

patients with MS. A, Quantitative summary of numbers of commensal-specific IgA ASC

detected in MS patients (n=6) and age-matched HC using the RePOOPulate human bacteria

template, Wilcoxon Test. B, Representative images of commensal-specific IgA ASC in HC vs.

MS. C-D, Comparison of quantification of microbiome IgA ASC detected in one MS patient

between healthy control human bacteria template, MS-associated template and MS non-

associated template. PBS-coated wells were used as a negative control for the assay.