# Lifelong evolution of autoreactive plasma cell numbers, affinity and anatomical location in arthritic K/BxN mice

Thibault Vanhoucke, Carlos Castrillon, Odile Richard-le Goff, Angga Perima, Matteo Broketa, Andrew D Griffiths, Patrick England, François Huetz, Pierre Bruhns

► To cite this version:

Thibault Vanhoucke, Carlos Castrillon, Odile Richard-le Goff, Angga Perima, Matteo Broketa, et al. Lifelong evolution of autoreactive plasma cell numbers, affinity and anatomical location in arthritic K/BxN mice. Arthritis & rheumatology, inPress, Online ahead of print. 10.1002/art.43436. pasteur-05393490

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*Running head: 20-month follow-up of autoreactive plasma cells in arthritic mice*

**Title:**

**Lifelong evolution of autoreactive plasma cell numbers, affinity and**
**anatomical location in arthritic K/BxN mice**

Thibault Vanhoucke, MSc<fnref n="1" />,<fnref n="2" />,§, Carlos Castrillon, PhD<fnref n="1" />,§, Odile Richard-Le Goff<fnref n="1" />,§, Angga Perima, PhD<fnref n="1" />,§, Matteo Broketa, PhD<fnref n="1" />,<fnref n="2" />, Andrew D. Griffiths, PhD<fnref n="3" />, Patrick England, PhD<fnref n="4" />, François Huetz, PhD<fnref n="1" /> and Pierre Bruhns, PhD<fnref n="1" />,*

§ these authors contributed equally.

Accepted Article

<footnote n="1">¹Institut Pasteur, Université de Paris, Unit of Antibodies in Therapy and Pathology, Inserm UMR1222, 75015 Paris, France.</footnote>

<footnote n="2">²Sorbonne Université, Collège Doctoral, Paris, France,</footnote>

<footnote n="3">³Laboratoire de Biochimie (LBC), ESPCI Paris, PSL Research University, CNRS UMR8231 Chimie Biologie Innovation, Paris, France.</footnote>

<footnote n="4">⁴Plateforme de Biophysique Moléculaire, Institut Pasteur, CNRS UMR3528, Paris, France.</footnote>

<footnote>* corresponding author: Pierre Bruhns, Unit of Antibodies in Therapy and Pathology, Institut Pasteur, 75015 Paris, France. Phone: +33-145688629. pierre.bruhns@pasteur.fr</footnote>
ABSTRACT

**Objective:** The spontaneous K/BxN mouse model of rheumatoid arthritis has been used extensively to study chronic inflammation, contribution of immune cells, and the primordial role of autoreactive antibodies in disease initiation and severity. Only the ubiquitous enzyme glucose-6-phosphate isomerase (GPI) is the target of IgG autoantibodies secreted by autoreactive plasma cells and plasmablasts in K/BxN mice. Strikingly, the appearance and evolution of these autoreactive IgG-secreting cells remain unstudied.

**Methods:** Here, we quantitatively and qualitatively investigated the plasmablast and plasma cell responses by measuring the affinity of their secreted antibody for GPI from single cells in cohorts of K/BxN mice from 3 to 87 weeks of age.

**Results:** Analysis of more than 36,000 individual IgG-secreting cells from spleen, popliteal lymph nodes, and bone marrow revealed high intercellular variability in affinity for GPI, with variations over 3 logs, with stable secretion rates over the life of the mice. Autoreactive IgG-secreting cells were detectable at 3-4 weeks and reached peak proportions of IgG-secreting cells at 35 weeks before stabilizing. High-affinity anti-GPI IgG-secreting cells appeared only transiently in the 6-9-weeks postnatal window, whereas low-affinity IgG-secreting cells represented more than 80% of the response at all time points. Serum anti-GPI IgG antibodies evolved in a similar kinetic fashion, peaking in proportion to total IgG and in affinity for GPI at 35 weeks.

**Conclusion:** Our results report the dynamic nature of the autoimmune B-cell response in the K/BxN model, revealing a transient phase of antibody affinity maturation early in disease that allows high-affinity antibody responses.

Accepted Article
INTRODUCTION

The hallmark antibodies in Rheumatoid arthritis (RA) are rheumatoid factors and
antibodies to post-translationally modified proteins e.g., citrullinated protein, detectable in 60-
70% of RA patients, but also antibodies to ubiquitously expressed enzymes like glucose-6-
phosphate-isomerase (GPI) antibodies in subgroups of RA patients¹. The presence of
autoantibodies in patients correlates with more severe disease course². Targeting the source of
antibody production, using anti-CD20 rituximab to deplete B cells³ or using anti-CD38
daratumumab to deplete plasma cells⁴, has proven efficacious in RA.

To understand the role of autoantibodies in RA, spontaneous mouse models of RA have
been developed, such as the K/BxN model⁵. In this model, arthritis arises spontaneously by the
production of autoantibodies targeting GPI that deposits on the articular cartilage. K/BxN mice
are C57BL/6 x non-obese diabetic (NOD) F1 mice which express the KRN transgenic T cell
receptor (TCR) that recognizes a peptide from GPI presented by the NOD-encoded I-Ag<sup>7</sup> MHC
class II molecule. This leads to a massive T cell and concomitant B cell activation, with high
quantities of anti-GPI autoantibodies produced, causing cartilage destruction and symptoms
resembling RA. Pathogenic IgG anti-GPI autoantibodies appear by 3 weeks of age<sup>6</sup>, accumulate
at high concentration in K/BxN serum (&gt;6 mg/mL)<sup>7</sup>.

This massive adaptive immune response against GPI in K/BxN mice relies on KRN
TCR-expressing T cell subsets e.g., T helper 17 (Th17) cells, T follicular helper (Tfh) cells and
dendritic cells that promote extensive polyclonal B cell activation during germinal center
reactions that start in the lymph nodes (LN) draining the distal joints<sup>8, 9, 10</sup>. Both T cell-
independent activation of marginal zone B cells by circulating GPI and T cell-dependent
activation of germinal center (GC) B cells by antigen-presenting cells contribute to the pool of
anti-GPI short-lived plasmablasts and long-lived plasma cells<sup>11</sup>. Initial descriptions of IgG-
secreting cells (IgG-SC) i.e., plasmablasts and plasma cells, in 8-week-old K/BxN mice

<figure><img src="image_2.png" /></figure>
suggested their very high proportion in lymph nodes and spleen but lower proportions in blood and bone marrow. Anti-CD20 B cell depletion experiments reduced their numbers 10-fold in lymph nodes and spleen, and 100-fold the circulating anti-GPI IgG titers, suggestive that CD20⁺ short-lived plasmablasts are largely predominant over CD20⁻ long-lived plasma cells in the K/BxN model¹². The dynamics of IgG-SC generation and anatomical distribution have, however, never been described in this extensively used mouse model of RA. The model has been considered to raise low-affinity anti-GPI antibody responses due to the strong extrafollicular B cell responses and short-lived plasma cell responses, but subnanomolar affinity mAbs could be isolated from ~8-week-old K/BxN mice⁶, indicating the generation of high-affinity anti-GPI B cells in this autoimmune model of RA.

Herein, we investigated over 20 months the dynamics of IgG-SC generation and their anatomical distribution among popliteal lymph nodes, spleen and bone marrow of K/BxN mice. Our data identifies a narrow window of time for autoreactive B cell expansion and affinity maturation, restricting affinity enhancement to 1 log on average over the lifetime of K/BxN mice.

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# MATERIAL AND METHODS

## Reagents

Mouse Glucose-6-Phosphate Isomerase (GPI; CSB-EP009717MO) was purchased from Cusabio. GPI was fluorescently labeled using AlexaFluor488 (AF488) NHS-Ester dye (Thermo Fisher Scientific #46403). Anti-GPI mAbs clones B10.8, D24.9, D24.4 and E4.6 were produced from hybridomas generated in house and purified by affinity chromatography using an AKTA pure FPLC instrument on a HiTrap Protein G column and desalted on a HiTrap desalting column (all Cytiva).

## Mice breeding

K/BxN and BxN mice were bred at Institut Pasteur (Paris, France) by crossing non obese diabetic (NOD; purchased from Charles River) mice with transgenic KRN mice or C57BL/6 wt mice, respectively. Wild-type BALB/c mice (purchased from Janvier labs). All animal care and experimentation were conducted in compliance with the guidelines and specific approval of the Committee for Ethics in Animal Experimentation (Institut Pasteur, Paris, France), and by the French Ministry of Research.

## Aqueous phase I: cell preparation for droplet encapsulation

Cells from spleen, popliteal lymph nodes and bone marrow from tibia and femur were collected and filtered through a 100-µm cell strainer before red blood cell lysis (BD BioSciences buffer #555899). Cell density was adjusted at 30.10⁶/mL in DropMap buffer (RPMI without phenol red (Gibco), 0.1% Pluronic F68 (Gibco), 25 mM HEPES pH 7.4 (Gibco), 5% KnockOut serum replacement (Thermo Fisher Scientific), 0.5% human serum albumin (Sigma Aldrich)) to achieve an average number of approximately 0.3 cell per droplet. For the generation of data points for the calibration curves, anti-GPI mAbs were diluted in DropMap buffer.

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## Aqueous phase II: beads and reagents preparation

Streptavidin-coated paramagnetic nanobeads (Ademtech, #03231) were incubated in DPBS without calcium and magnesium (Gibco) containing 1 µM CaptureSelect biotin anti-kappa light chain conjugate (Thermo Fisher Scientific) for 20 minutes at room temperature, washed and incubated in 5% Pluronic F68 solution (Thermo Fisher Scientific #P6866) for 20 minutes at room temperature. Nanobeads were incubated in DropMap buffer for 20 minutes at room temperature, washed in DPBS and resuspended in DropMap buffer containing 60 nM of AF488-labelled GPI (final in-droplet concentration: 30 nM) and 150 nM of an AF647-labelled rabbit anti-mouse IgG F(ab')2 (final in-droplet concentration: 75 nM) (Jackson ImmunoResearch #315-606-046).

## DropMap: material production, droplet encapsulation and data acquisition

The silicon wafer, the chip with the droplet chamber and the droplets were produced as we described previously¹⁴. Images of the droplet chamber were acquired on an inverted microscope with a motorized stage (Ti2-Eclipse, Nikon) and a high-speed camera (Orca Flash 4.0, Hamamatsu) at room temperature. every 7.5 min over a period of 37.5 min as reported¹⁴.

## Image analysis and calculation

Images were analyzed using a custom-made MATLAB script (MathWorks) to identify each droplet and its beadline. In each fluorescence channel and for each droplet, the pixel intensities of the beadline and the mean pixel intensities except the beadline (background fluorescence) were extracted. Droplet fluorescence relocation at each time point was calculated by dividing the intensity of the beadline by that of the background. Droplets showing between the first and last time point a minimum relocation >1.2 and a net difference >0.025, and a minimal slope of 0.004 were considered positive for IgG secretion. These droplets were visually assessed for the presence of a single cell or two cells (droplets with more than two cells were excluded), no

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droplet movement between image acquisitions, and the absence of fluorescence artefacts, as described¹⁴. The computed metrics of AF647 and AF488 fluorescence relocation over time were converted into IgG secretion rates and affinity for GPI by using calibration curves generated with a panel of monoclonal antibody of known affinities for GPI encapsulated in droplets at different concentration (Fig. 1C, Supplemental Fig. 1B).

## Anti-GPI ELISA

96-well plates (Costar) were coated with 5 µg/ml of rabbit GPI (Sigma Aldrich, #P9544) in carbonate/bicarbonate buffer (pH 9.6), blocked with PBS-Tween containing 2% Bovine Serum Albumin (BSA) (Roche), washed and incubated with K/BxN or BxN mice serum samples diluted 1:300 to 1:218,700. Bound antibodies were detected by addition of goat anti-mouse IgG Fc fragment HRP conjugated (Bethyl Laboratories, A90-131Ps) at 1:4,000, and revealed using OPD substrate (Sigma Aldrich, #P9187), stopped by addition of 2M H₂SO₄. Absorbance was recorded at 492 nm and 620 nm.

## Kinetics measurement by Surface Plasma Resonance (SPR)

For SPR analysis (Biacore™, Cityva, MA, USA), rabbit anti-mouse IgG Fcγ fragment specific antibody (Jackson ImmunoResearch, 315-005-046) were immobilized on Sensor CM5 chips (Cityva). IgG contained in K/BxN or BxN serum were captured during 300 second injections. Mouse GPI was injected at various concentrations (2000, 667, 222, 74, 25 nM) in 1X PBS + 0.01 % (v/v) Tween® 20 + 0.2 mg/mL BSA for 300 seconds. Flow paths were regenerated by two cycles of Glycine HCl 10mM pH5 and one cycle NaOH 10mM. Polyclonal affinity for mouse GPI and Rmax was determined using 1:1 ‘Steady state affinity’ modelling (Biacore T200 Evaluation software 3.1, Cytiva). Percentage of anti-GPI IgG antibodies was calculated with the following formula (M<sub>GPI</sub>, molar mass of GPI; M<sub>IgG</sub>, molar mass of IgG):
$$ \% = \frac{R_{max}}{\text{IgG capture level} \times \frac{M_{GPI}}{M_{IgG}}} \times 100 $$

## Flow cytometry

To identify anti-GPI-specific plasma cells and plasmablast by flow cytometry, single cell suspensions from spleen, popliteal lymph nodes and bone marrow were permeabilized and stained for B220, CD138 and AF488-labeled GPI. Control experiments used AF488-labeled ovalbumin instead of GPI and demonstrated no positive plasma cells. Flow cytometry acquisition was performed on a MacsQuant cytometer (Miltenyi) and analyzed on FlowJo software (BD Biosciences).

## K/BxN-passive arthritis (serum transfer experiment)

K/BxN serum was collected from the same mice that were used for DropMap experiments, the day of the sacrifice. One pool of serum was generated using serum from K/BxN mice aged 6-12 weeks and another from serum collected from K/BxN mice aged 72-83 weeks. As a negative control, a pool of serum was generated using serum from KRN transgenic C57BL/6 mice aged 8-14 weeks. Wild-type BALB/c mice (purchased from Janvier labs) were injected intraperitoneally with 200 µL serum on day 1 and 3. Ankle swelling was evaluated by ankle thickness measured separately on the right and left posterior paw using a caliper (Kroeplin S0247) from day 1 to day 12.

## Statistics

GraphPad Prism version 10.4.1 was used for all analyses. Data were not normalized or normalized prior to being analyzed. All data points present single-cell values unless otherwise stated. Samples were not pooled unless explicitly stated. Affinity and secretion data were determined to be neither normally distributed nor log-normally distributed according to the D'Agostino and Pearson, Anderson-Darling, and Kolmogorov-Smirnov tests. Paired or

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unpaired t-tests and Mann-Whitney tests were used for comparing samples or groups of samples. Kruskal-Wallis test with Dunn's test for multiple comparisons was used on aged-grouped affinity values from DropMap data in Supp. Fig. 2F. 2-way ANOVA with multiple comparison was used to determine significance for data from K/BxN passive arthritis experiments. Statistical significance was set to a P value of 0.05 or less.

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RESULTS

K/BxN mice develop arthritic symptoms from ~4-weeks of age and survive up to 85-120 weeks. Anti-GPI autoantibodies were detectable starting 3 weeks of age, increased rapidly until 10-11 weeks of age, and plateaued until 35 weeks of age before modestly but significantly reducing afterwards (Fig. 1A). We evaluated the evolution of this circulating anti-GPI antibody pool in terms of the specific fraction prevalence and affinity for GPI by surface plasmon resonance (SPR) measures on K/BxN mouse serum for 14 time points from week 3 to week 87. Serum anti-GPI IgG polyclonal affinities evolved from a very poor 4 µM at 3 weeks of age to their peak at 120 nM at week 11 and remained relatively stable until 87 weeks of age (Fig. 1B). The proportion of anti-GPI IgG autoantibodies among all IgG evolved in successive plateaus from 10-15% between week 3 and week 8, to 30-46% between week 11 and week 35, before reducing back to 15% between week 72 and week 87 (Fig. 1C). These data highlight three different phases of anti-GPI IgG in circulation and presumable B cell responses in parallel with: i) 1 log improvement of affinity for GPI (weeks 3-8); ii) increase in anti-GPI IgG proportion with 0.5 log affinity improvement (weeks 10-35); iii) reduction in anti-GPI IgG proportion with minimal variations in affinity (> 35 weeks). Analyzing rare samples of >100-week-old K/BxN mice confirmed this evolution with anti-GPI IgG proportion down to 1% at 113 weeks without significant loss in affinity compared to 35-week-old mice (Fig. 1D; a compilation of Figures 1B and 1C). Altogether these data suggest early life appearance of anti-GPI IgG⁺ plasmablasts and plasma cells that contribute to the circulating pool of anti-GPI IgG, with a rapid increase in cell numbers and affinity maturation of the B cell response up to ~10 weeks of age followed by a slower evolution until ~35 weeks of age.

To confirm this hypothesis on autoreactive plasma cell dynamics in the first weeks of
life of K/BxN mice, we analyzed plasmablast and plasma cells (defined as B220<sup>lo/-</sup> CD138<sup>hi</sup>)
and among them GPI-specific plasma cells by flow cytometry on permeabilized cells from

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spleen, popliteal lymph nodes (PLN), and bone marrow of K/BxN mice between 3 and 20 weeks of age. GPI-specific and non-specific plasmablast and plasma cell numbers increased rapidly in spleen and PLN until week 10, before decreasing by week 20 (Fig. 1E, Supp. Fig. 1A-B). In the bone marrow, numbers and abundance of GPI-specific plasma cells remained lower but steadily increased up to week 20 (Supp. Fig. 1A-B). To further understand what factors may drive these dynamics, we characterized T follicular helper (Tfh) cells (defined as $B_{220^{-}}^{CD4^{+}}$ PD-1$^{hi}$ CXCR5$^{hi}$) and germinal center (GC) B cells (defined as $B_{220^{+}}^{CD4^{-}}$ GL7$^{+}$ FAS$^{+}$) numbers and proportions by flow cytometry in the same timeframe. Tfh cell numbers and proportions among total T cells increased in spleen from 3 to 20 weeks, whereas in PLN they decreased after 10 weeks (Supp. Fig. 1C). In both organs, ≥85% of these Tfh cells expressed the KRN TCR ($v\beta6$), suggesting their involvement in anti-GPI autoreactive reactions (Supp. Fig. 1D). GC B cell numbers and proportions among total B cells also increased rapidly until week 10, before reducing or plateauing (Supp. Fig. 1E). The combined reduction of GPI-specific Tfh cells and GC B cells in spleen and PLN after 10 weeks of age may be responsible for the decrease in GPI-specific plasma cells and plasmablasts after week 10, and may also contribute to the lack of anti-GPI IgG affinity maturation after week 10 observed in polyclonal serum of K/BxN mice (Fig. 1B).

We sought to analyze the affinity maturation of the anti-GPI response in K/BxN mice by measuring over time the affinity for GPI of IgG secreted by $IgG^{+}$ plasmablasts and plasma cells, which here we collectively term IgG-secreting cells (IgG-SC). We thus adapted the single-cell bioassay in microfluidic droplets termed “DropMap” that we have described previously¹³, allowing a direct characterization of GPI-specific IgG-SC in terms of numbers, IgG secretion and affinity of their IgG for GPI. Cells from spleen, popliteal lymph nodes, and bone marrow of K/BxN mice were co-encapsulated with paramagnetic nanobeads and fluorescent bioassay reagents in droplets (Fig. 2A) before being immobilized within an observation chamber and

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imaged over 37.5 minutes by time-lapse fluorescence microscopy (Supp. Fig. 2A). The nanobeads are pre-coated with an anti-mouse kappa (κ)-light chain nanobody to capture secreted immunoglobulins onto the beads that align into a stick-like shape (termed beadline) under a magnetic field. This beadline acts as a physical surface for a fluorescent sandwich immunoassay revealing IgG,κ secretion by the cell inside the droplet and binding of that IgG,κ to GPI (Fig. 2B, Supp. Fig. 2B). Image analyses extract binding metrics that are converted using reference curves (Supp. Fig. 2C-D) into IgG secretion rates within a 4-800 molecules/s range and into affinity (K_D) for GPI within a 2.3-100 nM range. Therefore, hereafter IgG-SC secreting IgG interacting with GPI at a computed K_D <100 nM are considered as GPI-specific, with K_D values below 2.3 nM mathematically extrapolated.

In spleen and PLN, anti-GPI IgG-SC were detected from 4 and 3 weeks, respectively, to 87 weeks with affinities for GPI ranging from a few pM to 100 nM (mean affinity ~65 nM). Low-affinity IgG-SC (K_D ≥ 10 nM) predominated whereas high-affinity IgG-SC (K_D < 1 nM) could only be detected between 6 and 9 or 8 and 11 weeks of age in spleen and PLN, respectively (Fig. 2C-D). In the bone marrow, anti-GPI IgG-SC were detected starting 3 weeks of age with affinities for GPI ranging from 0.86 to 100 nM (mean affinity ~75 nM). Only low-affinity IgG-SC, but no high-affinity IgG-SC (except one at week 6) were detected in the bone marrow (Fig. 2E). Overall, anti-GPI IgG-SC appeared in spleen, PLN and bone marrow of K/BxN mice immediately before the onset of disease symptoms, and rapidly increased in numbers until a peak at week ~10 before decreasing to a plateau. The mean anti-GPI affinity of IgG-SC in all three compartments did not vary significantly from one time point to another over the lifetime of the mice (Supp. Fig. 2E), with at all time points >80% low-affinity IgG-SC and ≤2.3% high-affinity IgG-SC; the latter cells detectable only during a narrow timeframe, from 6 to 11 weeks (Supp. Fig. 3). However, when compared to other age groups, anti-GPI IgG-SC from the 6-12-week age group demonstrated significantly higher affinities than those from

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older age groups (Supp. Fig. 2F), which is suggestive of a temporal decline in affinities among anti-GPI IgG-SCs in the popliteal lymph nodes and spleen, and to a lesser extent in the bone marrow.

The poor affinities of IgG secreted by IgG-SC for GPI might be compensated by the large fraction of anti-GPI IgG-SC among all IgG-SC that reached >20% on average with peaks at ~50% (Supp. Fig. 4). By quantifying using DropMap the secretion rate of IgG by every IgG-SC analyzed in this study, we measured secretion rates ranging from 3 to 336 IgG/s (mean 33 IgG/s) in spleen, from 4 to 510 IgG/s (mean 33 IgG/s) in PLN and from 3 to 284 IgG/s (mean 21 IgG/s) in bone marrow (Fig. 3A-C). Peak secretion rates were measured between 6 and 11 weeks of age with outliers above 200 IgG/s representing less than 3% of all IgG-SC (Supp. Fig. 5). No correlation between secretion rates and affinity for GPI was identified (Supp. Fig. 6A-C), with non-GPI-specific IgG-SC measured at higher IgG secretion rates than GPI-specific IgG-SC in PLN and bone marrow, but not significantly in spleen (Supp. Fig. 6D-F). The increase in anti-GPI IgG content over time in circulation is therefore not attributable to enhanced IgG secretion, but uniquely to the increasing fraction and maturation of autoreactive IgG-SC.

In order to determine whether the 6-11 week period in the life of K/BxN mice demonstrating the presence of high-affinity anti-GPI IgG-SC leads to the formation of a circulating pool of antibodies with higher pro-arthritic properties than a period without these high-affinity anti-GPI IgG-SC, we collected and pooled serum from K/BxN mice aged 6-12 weeks and 72-83 weeks, as well as from control KRN mice. As expected, K/BxN serum transfer, but not KRN serum transfer, led to clinical signs of inflammatory rheumatoid arthritis, as measured by increased ankle thickness. Notably, serum from K/BxN mice aged 6-12 weeks
induced significantly more severe and prolonged ankle swelling than serum from K/BxN mice
aged 72–83 weeks (see Fig. 3D).
DISCUSSION

Our study describes the dynamics of IgG-SC over 20 months in K/BxN mice, starting one week before disease onset. It identifies a 6-week time window that supports rapid anti-GPI IgG-SC expansion and antibody affinity maturation between 4 and 10 weeks of age, followed by slow IgG-SC response improvement until 35 weeks of age before plateauing and declining. At the peak of the anti-GPI IgG-SC response, ~50% of IgG-SC in the PLN and ~35% of IgG-SC in the spleen and bone marrow, and ~45% of circulating IgG, were specific for GPI. Because anti-GPI autoantibodies are heavily skewed towards the IgG1 isotype$^{6,7}$, this fraction may be even higher among IgG1-SC. The secretion rate of anti-GPI IgG-SC remained nevertheless constant in the three anatomical locations over 87 weeks, with peaks of single cell-secretion rates correlating with the early and rapid IgG-SC expansion phase.

Using the DropMap droplet microfluidic approach¹³, we identified herein IgG-SC without the use of any surface marker, but only on their capability to secrete IgG and among them anti-GPI IgG. Flow cytometry data on permeabilized CD138<sup>hi</sup> plasmablasts and plasma cells confirmed the dynamics of anti-GPI IgG-SC that we identified through DropMap in the popliteal lymph nodes, spleen and bone marrow. A rapid expansion phase of IgG-SC correlated with that of KRN TCR-expressing Tfh cells and of GC B cells, confirming earlier observation on the primordial role of Tfh cells for B cell maturation into plasmablasts and plasma cells⁹. Plasmablasts have been identified as major cellular players in the K/BxN model as anti-CD20 B cell depletion experiments, which target plasmablasts but not long-lived plasma cells, reduced circulating anti-GPI IgG titers 10-fold over an 8-week treatment¹². GPI-specific IgG-secreting plasmablasts and plasma cells accounted for a large fraction of total IgG-SC, with a minimum of 20% after 20 weeks in spleen, PLN and bone marrow, even at later time points. These results indicate a chronic generation of autoreactive B cells throughout the lifetime of K/BxN mice.

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Anti-GPI IgG titers and IgG-SC numbers did not evolve linearly in K/BxN mice. As reported earlier⁶, anti-GPI antibody titers increased rapidly after 3 weeks of age, plateaued and slowly decreased. Anti-GPI IgG-SC followed the same kinetics with appearance in spleen, PLN and bone marrow at 3-4 weeks of age, peaking between 6 and 12 weeks of age and decreasing in numbers thereafter. The median affinities, however, did not vary in any of the three anatomical compartments, supporting the very large predominance of low-affinity autoreactive IgG-SC (probably essentially plasmablasts) in this spontaneous RA model. Analyzing data per age group (6-12; 23-35; 54-87 weeks of age), we found significant temporal decline of the affinities of IgG secreted by anti-GPI IgG-SC in the popliteal lymph nodes and spleen, and to a lesser extent in the bone marrow. The highest proportion of anti-GPI IgG-SC were observed in draining lymph nodes, though their largest reservoir was the spleen due to organ size differences. The affinity for GPI of the IgG secreted by these IgG-SC also evolved similarly, with detectable high-affinity (K_D <1 nM) antibodies only between 6 and 11 weeks of age, restricted to spleen and PLN. The bone marrow appeared unable to host high-affinity IgG-SC (at least detectable numbers thereof) which might be linked to the control of long-lived plasma cell survival by Foxp3⁺ regulatory T cells in this model¹⁴. This peak of medium- and high-affinity IgG-SC correlates with joint inflammation, which reduces in this model after 10-weeks of age to be followed by cartilage destruction and bone erosion, indicative of a modification of the immune response. The fact that serum collected during the peak of high-affinity IgG-SC was significantly more pro-arthritic than serum collected at late time points may be a consequence of this affinity maturation burst.

Our study is limited by its focus on affinity measurement throughput. A DropMap assay captures only a small fraction of the IgG-SC within a given organ. Indeed, one acquisition corresponds to an average of 60,000 to 80,000 droplets, among which only 20,000 contain one or two cells and is thus taken into consideration in our analyses. Assuming the spleen of 3-

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week-old mice contains roughly 30x10⁶ cells, we estimate that we screened only 0.07% of the
spleen per DropMap. In addition, the in-droplet assay was designed to capture κ-light chain
antibodies onto the beadline, making it impossible to detect any λ-light chain anti-GPI
autoantibodies. As mice produce 95% of κ-light chain antibodies, we nevertheless assume that
only rare autoreactive IgG-SC might have been overseen in our work. Finally, the detection
limit of anti-GPI IgG affinity being 100nM in DropMap assays, lower affinity interactions were
not considered, suggesting that the proportion of low-affinity anti-GPI IgG-SC may be even
higher than we report herein.

This study provides a direct *ex vivo* characterization of autoreactive IgG-SC during
initiation and development of arthritis in K/BxN mice. Our results report on the dynamic nature
of the autoimmune B cell response in this model that highlights - within a restricted time
window - an escape from tolerance leading to antibody affinity maturation.

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ACKNOWLEDGMENTS

We would like to thank our colleagues from the Institut Pasteur, Paris, France for their help and advice: François Traincard (Antibody Engineering Platform) for selection of anti-GPI hybridomas; Bertrand Raynal, Sébastien Brulé and Sylviane Hoos for assistance with affinity measurements (Molecular Biophysics Core Facility) and Henna Maulaboksh for administrative help. No AI-based solution has been used to write this manuscript. This work benefited from the technical contribution of the joint service unit CNRS UAR 3750. We acknowledge the support of Biomaterials and Microfluidics Platform and FabLab at Institut Pasteur.

FUNDING

Work in the Bruhns' lab was supported by the Institut Pasteur, Institut National de la Santé et de la Recherche Médicale (Inserm), Fondation pour la Recherche Médicale, Paris, France (Programme Equipe FRM grant EQU202203014631 to PB), Agence Nationale de la Recherche (grant ANR-22-PEBI-0009 PEPR ACCREDIA to PB) and from the CAPNET (Comité ad-hoc de pilotage national des essais thérapeutiques et autres recherches, French government) MEMO-VOC and MEMO-BREAK. TV is the recipient of a PhD fellowship from École polytechnique, Palaiseau, France (AMX fellowship 2023–2026). MB was the recipient of a CIFRE PhD fellowship in collaboration with Evextabio, Paris, France. CC acknowledges financial support from CONCYTEC, Peru.

AUTHOR CONTRIBUTIONS

**Thibault Vanhoucke**: Investigation; Software; Formal analysis; Writing.
**Carlos Castrillon**: Investigation; Formal analysis; Writing - review & editing.
**Odile Richard-Le Goff**: Investigation; Formal analysis; Writing - review & editing.
**Angga Perima**: Investigation;

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Software; Formal analysis; Writing - review & editing. **Matteo Broketa**: Software; Formal analysis; Writing - review & editing. **Andrew D. Griffiths**: Formal analysis; Supervision; Writing - review & editing. **Patrick England**: Formal analysis; Writing - review & editing. **François Huetz**: Formal analysis, Supervision; Writing - review & editing. **Pierre Bruhns**: Formal analysis; Resources; Conceptualization; Writing.

## COMPETING INTERESTS

Unrelated to the submitted work, PB received consulting fees from Regeneron Pharmaceuticals, NY, USA, and from Merida Biosciences, MA, USA, without any relation to the content of this manuscript. The other authors declare no competing interests.

## DATA AND MATERIALS AVAILABILITY

Further information and requests for resources, reagents and material should be directed to and will be fulfilled by the Lead Contact, Pierre Bruhns [bruhns@pasteur.fr](mailto:bruhns@pasteur.fr)
<figure><img src="image_3.png" /></figure>

REFERENCES

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7. Mancardi, D.A., Jonsson, F., Iannascoli, B., Khun, H., Van Rooijen, N., Huerre, M. et al. Cutting Edge: The murine high-affinity IgG receptor FcgammaRIV is sufficient for autoantibody-induced arthritis. J Immunol 2011;186(4):1899-1903.

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14. Jang, E., Cho, W.S., Cho, M.L., Park, H.J., Oh, H.J., Kang, S.M. et al. Foxp3+ regulatory T cells control humoral autoimmunity by suppressing the development of long-lived plasma cells. J Immunol 2011;186(3):1546-1553.

Accepted Article
FIGURE LEGENDS

**Fig. 1. Dynamics of circulating anti-GPI antibodies in K/BxN. (A)** ED50 computed from anti-GPI ELISA on serum taken at indicated ages from K/BxN mice. Values outside of dilution range were indicated as either <300 or >218,700. (B-C) Surface Plasmon Resonance measures of (B) average anti-GPI affinity and (C) proportion of anti-GPI IgG autoantibodies among circulating IgG on serum taken at indicated ages from K/BxN mice. (D) Kinetic evolution of average anti-GPI affinity and autoantibody proportion among circulating IgG, with mean ±SEM indicated. The orange arrow indicates the trajectory taken for average affinity and autoantibody proportion over time from 3 (start of line) to 113 weeks (arrowhead). (E) Kinetic evolution of the number of anti-GPI $B_{220}^{+} CD_{38}^{+}$ plasma cells/plasmablasts in indicated tissues. (A, B, C) Consecutive timepoints were compared using a Mann-Whitney test. *: p<0.05, **: p<0.01.

**Fig. 2. Kinetic evolution of IgG autoantibody-secreting cell affinity for GPI in spleen, PLN and bone marrow. (A)** Schematic showing the inputs and outputs of microfluidic encapsulation. Cells (top) are flowed in parallel with bioassay components (GPI-AF488, magnetic beads coated with anti-κ VHH, and anti-IgG $F(ab')_2$-AF647) on the droplet nozzle. Oil flow closes the collected aqueous phases (cell component and bioassay component) into a water-in-oil droplet. The column of droplets depicts the potential outcomes of cell encapsulation. (B) Schematic showing the composition of the DropMap assay and potential changes to the assay as it progresses. In the absence of a cell (outer left) or with a non-IgG-SC (inner left), there is no change in GPI (green) or anti-IgG $F(ab')_2$ (red) fluorescence distributions over time. A non-GPI specific IgG-SC (inner right) in the droplet causes anti-IgG $F(ab')_2$ fluorescence to relocate to the beadline over time. A GPI-specific IgG-SC (outer right) causes both GPI and anti-IgG $F(ab')_2$ fluorescence to relocate to the beadline. (C-E) IgG-SC harvested

Accepted Article
Accepted Article

from the spleen (C), the popliteal lymph nodes (D) and bone marrow (E) from 3 to 87-week-
old mice and characterized using DropMap for anti-GPI affinity. Horizontal red lines represent
the mean affinity for each timepoint. Affinity values better than 10 pM were plotted at 10 pM.
The number of independent DropMap experiments for each timepoint is indicated on top of the
graph. Colored boxes next to the y-axis indicate low (blue), medium (yellow) and high (orange)
affinity, separated by dotted horizontal lines.

**Fig. 3. Kinetic evolution of IgG secretion from IgG-secreting cells in spleen, PLN and bone marrow, and evolution of pro-arthritic properties of K/BxN serum.** (A-C) IgG-SC harvested from the spleen (A), the popliteal lymph nodes (B) and bone marrow (C) from 3 to 87-week-old mice and characterized using DropMap for IgG secretion (IgG molecules/s). Horizontal red lines represent the mean secretion rate for each timepoint. The number of independent DropMap experiments for each timepoint is indicated on top of the graph. Colored boxes next to the y-axis indicate low (blue), medium (yellow) and high (orange) secretion rates, separated by dotted horizontal lines. (D) K/BxN serum transfer experiment in wild-type BALB/c mice using serum collected from 6-12- or 72-83-week-old K/BxN mice (n=6) or control KRN mice (n=3). “6-12w serum” curves were compared two-by-two with “72-83w serum” curves using a 2-way ANOVA multiple comparison test. ****: p<0.0001.

**Supporting Information**

Supinfo1
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Accepted
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