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Microchimeric cells promote production of rheumatoid
arthritis-specific autoantibodies

Marie Hemon, Mathilde Giassi, Yoan Ghaffar, Marielle Martin, Jean Roudier,

Isabelle Auger, Nathalie C Lambert

► To cite this version:

Marie Hemon, Mathilde Giassi, Yoan Ghaffar, Marielle Martin, Jean Roudier, et al. Microchimeric cells promote production of rheumatoid arthritis-specific autoantibodies. Journal of Autoimmunity, 2024, 146, pp.103238. 10.1016/j.jaut.2024.103238. hal-04798224

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https://amu.hal.science/hal-04798224v1

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# Microchimeric cells promote production of rheumatoid arthritis-specific autoantibodies

Marie Hemon<sup>a, b</sup>, Mathilde Giassi<sup>a</sup>, Yoan Ghaffar<sup>a</sup>, Marielle Martin<sup>a</sup>, Jean Roudier<sup>a, c</sup>, Isabelle Auger<sup>a</sup>, Nathalie C. Lambert<sup>a, *</sup>

## ARTICLE INFO

**Keywords:**

Microchimerism

Rheumatoid arthritis

ACPAs

Hapten/carrier model

## ABSTRACT

**Background:** Women are more likely to develop autoimmune diseases than men. Contribution from microchimerism (Mc) has been proposed, as women naturally acquire Mc from more sources than men because of pregnancy. Women with Rheumatoid Arthritis (RA) who lack RA-associated HLA alleles have been found to harbor Mc with RA-associated HLA alleles in higher amounts than healthy women in prior work. However, an immunological impact of Mc remains to be elucidated.

**Objectives:** To test the hypothesis that Mc with RA-risk associated HLA alleles can result in the production of RA-associated autoantibodies, when host genetic risk is absent.

**Methods:** DBA/2 mice are unable to produce RA-specific anti-citrullinated autoantibodies (ACPAs) after immunization with the enzyme peptidyl arginine deiminase (PAD) in a previously developed model. DBA/2 females were mated with C57BL/6 males humanized to express HLA-DR4, which is associated with RA-risk and production of ACPAs, to evaluate DR4+ fetal Mc contribution. Next, DBA/2 females born of heterozygous DR4+/− mothers were evaluated for DR4+ Mc of maternal or littermate origin. Finally, DBA/2 females from DR4+/− mothers were crossed with DR4+ males, to evaluate the contribution of any Mc source to ACPA production.

**Results:** After PAD immunization, between 20 % and 43 % of DBA/2 females (otherwise unable to produce ACPAs) had detectable ACPAs (CCP2 kit) after exposure to sources of Mc with RA-associated HLA alleles, compared to 0 % of unmated/unexposed DBA/2 females. Further the microchimeric origin of the autoantibodies was confirmed by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response.

**Conclusion:** Our study demonstrates that Mc cells can produce “autoantibodies” and points to a role of Mc in the biology of autoimmune diseases, including RA.

## 1. Conflict-of-interest statement

The authors have declared that no conflict of interest exists.

## 2. Introduction

Microchimerism (Mc) refers to the presence of a small number of cells (or DNA) from an individual genetically different from the host. A natural way to acquire Mc is during pregnancy [1]. A bidirectional transfer of cells between mother and fetus or between fetuses in twin pregnancies happens. Maternal- and fetal-origin Mc (MMc and FMc), as well as cells from twins, can persist for decades in blood and tissues from

their hosts [2–4]. Although we all are “microchimera,” as we all receive MMc, women naturally acquire Mc from more sources than men because of pregnancy [5].

It has been long hypothesized that this could be one of the reasons women are more often affected than men by “auto”-immune diseases and that microchimeric cells could be contributing to the pathogenesis of these diseases [6].

Rheumatoid arthritis (RA) is a chronic destructive joint disease whose origin is unknown with i) a female:male ratio of 3:1, ii) a strong genetic background represented by Human Leukocyte Antigens (HLA) DRB1 alleles (DRB1*01:01, *04:01, *04:04, *04:05, 04:08, *10:01) coding for a common five-amino-acid motif, the so-called Shared

<footnote><sup>a</sup> INSERM UMRs 1097 Arthrites Autoimmunes, Aix Marseille Université, Marseille, France</footnote>

<footnote><sup>b</sup> Arthritis R&D, Neuilly-sur-Seine, France</footnote>

<footnote><sup>c</sup> Rheumatology department, Assistance Publique des Hôpitaux de Marseille (AP-HM), Marseille France</footnote>

<footnote>* Corresponding author. INSERM UMRs1097 Arthrites Autoimmunes 163 avenue de Luminy, case 939 13288 Marseille, Cedex 09, France.
E-mail address: nathalie.lambert@inserm.fr (N.C. Lambert).</footnote>
Epitope (SE), in position 70–74 of the DRβ1 chain of the HLA-DR molecule [7], and iii) anti-citrullinated protein autoantibodies (ACPAs) appearing several years before the onset of the disease, allowing to facilitating the diagnosis of 70 % of patients [8].

We, and others, previously showed that women with RA who lack RA-associated HLA-DRB1\*01 or DRB1\*04 alleles had significantly higher levels of Mc carrying these alleles than matched controls [9]. Importantly, no difference in Mc was observed for alleles (HLA-DRB1\*15 and DQB1\*02) not associated with RA [9]. Another group confirmed our study and stepped beyond by developing SE-specific Q-PCR assays to specifically quantify SE positive Mc in SE negative patients [10]. In agreement with this hypothesis, Cruz et al. found in a large case/control study that the risk of developing RA was increased among women who had given birth to a child carrying RA-associated HLA-DRB1 alleles [11].

However, it remains to be proven that Mc cells contribute to immune functions in RA despite their relatively small number. An appropriate mouse model is needed to fully confirm this hypothesis.

It is strongly suggested that the ACPA response has a central role in disease-initiating processes and could drive chronicity of inflammation [12]. Protein citrullination is one of the post-translational modifications where peptidyl-arginine residues of the target protein(s) are converted to peptidyl-citrulline by enzymes called peptidyl arginine deiminases (PADs) [13]. Citrullination is a common physiological phenomenon, but the emergence of ACPAs is a unique feature of RA. Our group has shown that the ACPA production does not follow the classical immunological model, but rather a “Hapten/Carrier” model (for review [14]). When immunizing mice with the PAD enzyme, PAD peptides are presented by B cells to CD4⁺ T cells, which helps the production of anti-PAD antibodies, but also to any citrullinated protein (Hapten) bound by PAD (Carrier) [15]. Interestingly, the ACPA production in this recently described mouse model is MHC-restricted [15]. Thus, C3H mice expressing an I-Eβk chain homologous to the RA susceptible HLA-DRB1\*04:01 allele [15], or transgenic mice expressing RA-associated HLA-DRB1\*04:01 (KO/KI\*04:01), or HLA-DRB1\*04:04 (KO/KI\*04:04) alleles [16] are able to produce ACPAs. On the other hand DBA/2 mice expressing an I-Eβd chain, which is homologous to the “non-susceptible” HLA-DRB1\*04:02 allele, fail to develop ACPAs [15].

MHC restriction is used here to demonstrate that MHC susceptibility alleles transferred through fetal, maternal and/or littermate Mc (FMc, MMc and/or LMc) can contribute to ACPA's production after PAD immunization of non-susceptible host mice. We used DBA/2 mice, “unable” to produce ACPAs, i) either crossed with transgenic KO/KI\*04:04 males to test the contribution of fetal DRB1\*04:04 Mc ii) or born of heterozygous KO/KI\*04:04 mothers to test the contribution of maternal and littermate DRB1\*04:04 Mc, as both sources are undistinguishable in such a crossing [17] iii) or born of heterozygous KO/KI\*04:04 mothers and crossed with KO/KI\*04:01 males, to test the contribution of any Mc source (maternal and littermate DRB1\*04:04 Mc and fetal DRB1\*04:01 Mc).

Here we show that DBA/2 mice for whom DR4 positive chimeric cells have been transferred during gestation and persist post-delivery, generate ACPA response after PAD immunization while unmated DBA/2 mice are “unable” to produce these antibodies. We further confirm the microchimeric origin of the antibodies in the ACPA response by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response.

# 3. Material and methods

## 3.1. Ethical authorizations

Mice were housed at the Luminy INSERM Institute, Marseille (A1301303). All experiments were approved by the animal ethics committee (APAFIS N° 3266-201512181131807v4; N°0300703; N°10065 and N°22865-2020112517402887 v1). All animal care and

experimental procedures were performed in agreement with the Animal Ethics Committee of Marseille and the Ministère de l'Enseignement Supérieur et de la Recherche.

## 3.2. Mouse strains

DBA/2JRccHsd (DBA/2) mice were purchased from Envigo Laboratories. Major Histocompatibility Complex (MHC) haplotype antigens of DBA/2 mice are H-2<sup>d</sup>.

C57BL/6NRj (C57BL/6) mice were purchased from Janvier Labs, Le Genest- Saint-Isle, France. MHC haplotype antigens of C57BL/6 mice are H-2<sup>b</sup>.

Knock-out/knock-in (KO/KI) C57BL/6NRj mice were made by replacing I-Eβ genomic DNA (KO) with gene fragments containing HLA-DRA and HLA-DRB1\*04:04 or HLA-DRB1\*04:01 (KI) (H-2<sup>b</sup>/<sup>b</sup> DR404<sup>+/-</sup> or H-2<sup>b</sup>/<sup>b</sup> DR401<sup>+/-</sup>) as previously described [16] and abbreviated KO/KI\*04:04 or KO/KI\*04:01 (CIPHE, Marseille, France).

## 3.3. Crosses to obtain sources of pregnancy-derived DR4 positive Mc in DBA/2 females

DBA/2 (H-2<sup>d</sup>/<sup>d</sup>) females were mated with homozygous KO/KI\*04:04 (H-2<sup>b</sup>/<sup>b</sup> DR404<sup>+/-</sup>) males (Fig. 1A). Up to three gestations were performed to test whether this would increase the possibility to DBA/2 hosts to acquire more fetal DR404<sup>+/-</sup> Mc. Such DBA/2 females were only exposed to DR404<sup>+/-</sup> FMc.

Mouse characteristics (number of pups, age at delivery) are detailed in Suppl. Table S1).

Heterozygous F1 females (H-2<sup>d</sup>/<sup>b</sup> DR404<sup>+/-</sup>) obtained from the cross described in Fig. 1A were kept to be mated with DBA/2 (H-2<sup>d</sup>/<sup>d</sup>) males. From this cross, we could obtain either DBA/2 offspring (H-2<sup>d</sup>/<sup>d</sup> DR404<sup>-/-</sup>) or heterozygous (H-2<sup>b</sup>/<sup>d</sup> DR404<sup>+/-</sup>) offspring (Fig. 1B). Only DBA/2 female offspring (H-2<sup>d</sup>/<sup>d</sup> DR404<sup>-/-</sup>) were kept to be tested. Such DBA/2 females were only exposed to maternal and littermate DR404<sup>+/-</sup> Mc.

To obtain mice exposed to all three Mc sources, DBA/2 female offspring (H-2<sup>d</sup>/<sup>d</sup> DR404<sup>-/-</sup>) obtained as described above (Fig. 1B) were mated with C57BL/6 homozygous KO/KI\*04:01 (H-2<sup>b</sup>/<sup>b</sup> DR401<sup>+/-</sup>) males (Fig. 1C). Up to two gestations were performed to test whether acquisition of fetal DR401<sup>+/-</sup> Mc was higher compared to one gestation in such mice. Characteristics of mice are detailed in Suppl. Table S2).

## 3.4. Immunization protocols

Mice were immunized subcutaneously, as previously described [15, 16], with 100 µg of murine PAD2 (mPAD2) or injected as a control with phosphate buffered saline (PBS), in Freund's complete adjuvant (CFA). Three booster injections with 100 µg of mPAD2 or PBS in Freund's incomplete adjuvant (IFA) were given subcutaneously 15, 30 and 45 days later. Characteristics of mPAD2 immunized mice and PBS controls are described in Suppl. Table S3.

Murine PAD2 (mPAD2) was purchased from Proteogenix, Schiltigheim, France. Purity of recombinant mPAD2 protein (>90 %) was analyzed on SDS-PAGE gel using the GelAnalyzer software by Proteogenix. The absence of mPAD2 autocitrullination was checked as previously described [15,16].

## 3.5. Blood and plasma collection

Mice were anesthetized 5 min with isoflurane prior to blood draw in a citrate tube from the mandibular vein. Mice were serologically followed for ACPA production every two weeks after the first immunization until euthanasia at day 105–120 post first immunization. At euthanasia mouse blood was drawn for Mc research. Blood was centrifuged at 1500 rpm to collect plasma in the supernatant, used for serology as described below. White blood cells in the pellet were collected for
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Crosses to obtain DR4⁺ microchimerism (Mc) in DBA/2 females. (A) fetal contribution: DBA/2 (H-2<sup>d/d</sup>) females (thick black circle) were mated with a homozygous KO/KI*04:04 (H-2<sup>b/b</sup> DR404<sup>+/-</sup>) male and could be tested after one, two or three gestations for fetal DR4 Mc. (B) maternal and littermate contribution: Heterozygous F1 females (H-2<sup>d/b</sup> DR404<sup>+/-</sup>) obtained from the first cross described were kept to be mated with DBA/2 (H-2<sup>d/d</sup>) males to obtain DBA/2 (H-2<sup>d/d</sup> DR404<sup>-/-</sup>) and heterozygous D2B6F1 (H-2<sup>b/d</sup> DR404<sup>+/-</sup>) offspring. Only DBA/2 females (H-2<sup>d/d</sup> DR404<sup>-/-</sup>) can be tested for maternal DR404 Mc. It is to note that these females receive also littermate DR404 Mc, which is here undistinguishable from maternal Mc. (C) triple contribution: DBA/2 females (H-2<sup>d/d</sup> DR404<sup>-/-</sup>) which have received maternal and littermate Mc were kept to be mated with C57BL/6 homozygous KO/KI*04:01 (H2<sup>b/b</sup> DR401<sup>+/-</sup>) males to receive additional DR401 fetal Mc, thus having a triple contribution of DR4 Mc.</figcaption></figure>

DNA extraction and chimerism quantification as described below.

### 3.6. Detection of anti-mPAD2 antibodies by ELISA

Enzyme-linked immunosorbent assay (ELISA) to detect IgG anti-mPAD2 in plasma from immunized mice have been detailed elsewhere [15,16]. Briefly, plates were coated with 0.5 µg of mPAD2 and blocked with bovine serum albumin (BSA). Plasma from mice obtained at 15-, 30-, 45-, 75-, 105- and 120-days post first immunization were diluted to 1/40 and incubated with mPAD2. After washing with PBS, anti-mouse IgG-peroxidase was added. After washing with PBS, tetramethyl benzidine (TMB) was added. Optical density (OD) was read at 405 nm. Background OD was obtained by adding each plasma to a well without mPAD2. A positive plasma was defined by a [test OD/background OD] ratio higher than two.

### 3.7. Detection of ACPAs with anti-cyclic citrullinated peptides 2nd generation kit (CCP2 kit)

CCP2 kits were purchased from SVAR, Malmö, Sweden. Plasma from mice obtained at 60-, 75-, 90- and 105-days post immunization were diluted at 1/5 for all DBA/2 mice tested (unmated or mated) and at 1/20 for positive control mice (KO/KI*04:01 and *04:04) and incubated with cyclic citrullinated peptides. After washing with PBS, anti-mouse IgG-peroxidase was added. After washing, TMB was added. Reaction was stopped by sulfuric acid. OD was read at 450 nm.

Each OD value was corrected by subtracting the average OD obtained in the plasma-free wells. Mean background OD was obtained using plasma from mice injected with PBS (negative control). A plasma was arbitrarily defined as positive when a [test signal/mean background signal] ratio was greater or equal to four.

### 3.8. Detection of ACPAs with PepStarTM peptide microarrays

Using the PepStarTM peptide microarray technique (JPT Peptide Technologies, Berlin, Germany), Dr. Auger previously designed arrays to specifically identify ACPAs in mouse plasma [18]. The array comprises 169 15-mers from the alpha and beta chain of fibrinogen, vimentin, histon 4, enolase, proteoglycan, filaggrin, ebna 2 and collagen as

previously detailed [18]. Thirty-three arginine-containing peptides and their 136 citrulline-substituted variants were synthesized on cellulose membranes using SPOTTM synthesis technology. Details on peptide immobilization and quality controls of the arrays are detailed in Ref. [18]. All peptides were deposited (10–15 mol) in three identical subarrays per block/slide enabling analysis of assay homogeneity and reliability of the results.

Plasma profiling of 20 samples of DBA/2 female mice was realized. The 20 samples included unmated PBS control mice (N = 4), unmated mPAD2 immunized mice (N = 6), exposed to FMc mPAD2 immunized mice (N = 5) and exposed to all three Mc source mPAD2 immunized mice (N = 5). In parallel and as positive controls, samples from mPAD2 immunized KO/KI*04:01 (N = 5) and from KO/KI*04:04 (N = 5) female mice were tested. All plasma samples were diluted 1:200 in blocking buffer (3 % BSA, 0.1 % Tween 20 in TBS-Buffer). Plasma from a blood draw at 105 days post first immunization for DBA/2 mice and 75 days for C57BL/6 KO/KI mice, were incubated, washed and revealed on multiwell microarray slides as previously detailed [18] and for each spot, mean signal intensity was extracted. For each mouse strain (DBA/2 or C57BL6 KO/KI), mean background signal intensity was obtained for every peptide using all the plasma from mice immunized with PBS. A positive plasma was defined by a [mean test signal/mean background signal] ratio higher than three.

### 3.9. Detection and quantification of IgG<sub>2c</sub>, a C57BL/6 strain-specific immunoglobulin in ACPA response

The C57BL/6 Mouse Immunoglobulin Panel (Southern Biotech, Birmingham, USA) was used according to manufacturer recommendations as standards for quantitative studies of C57BL/6 mouse IgG<sub>2c</sub> in plasma samples. Six serial half dilutions, starting from 12.5 ng/mL of purified murine IgG<sub>2c</sub> isotypes were realized to construct the standard curve of reference (Suppl. Fig. S1) to calculate the concentration of IgG<sub>2c</sub> in plasma samples tested on CCP2 kits (see ACPA detection with CCP2 kit) where anti-mouse IgG-peroxidase was replaced by the secondary goat anti-mouse IgG<sub>2c</sub>-HRP antibody.

Of note, IgG<sub>2c</sub> is an isotype expressed by C57BL/6 mice and not by DBA/2 strain [19,20], and consequently expressed by KO/KI*04 mice constructed on a C57BL/6 strain. However, this is not the most
represented isotype among the different immunoglobulins (IgG1, 2b, 3) with values comprised between 5 % and 10 % of the total IgG response [21]. Thus, to detect whether microchimeric cells (with a C57BL/6 ge- netic background) participate in the production of anti-CCP antibodies, we used plasma more concentrated (dilution to the half) than for the analysis of KO/KI\*04 (1/250th). The final calculation of IgG2c con- centration (in ng/mL of plasma) took into account the dilution factor.

### 3.10. IgG2c/IgG2a genotyping

As the inheritance of mouse *IgG2a/2c* is on chromosome 12 and in- heritance of *H-2* is on chromosome 17, thus independent, a DBA/2 offspring (H-2<sup>d/d</sup> DR404<sup>-/-</sup>), born of a hemizygous H-2<sup>d/b</sup> DR404<sup>+/−</sup> mother and a DBA/2 male, has one in two chance of inheriting the maternal IgG2c gene. We thus designed PCR assays to specifically ge- notype DBA/2 mice born of hemizygous IgG2a/2c H-2<sup>d/b</sup> DR404<sup>+/−</sup> mothers (Ex Fig. 1B and C). The *IgG2c* sequence was found in Ensembl database (http://www.ensembl.org/index.html) as detailed in Zhang et al. [20], the *IgG2a* sequence is from Morgado et al. [19]. Primers and probes were synthesized by TIB MolBiol (Berlin, Germany) and were respectively for IgG2a and IgG2c: Mus-IgG2a-forward CATgTA-CAgCAAgCTgAgAgTgg; Mus-IgG2a-reverse CgggAgAAgCTCTTAgTCg TgT and Mus-IgG2a probe 6FAM-CAggAgTAgCTATTTCTTTCCACC-CAgTTCTTCT—TMR; Mus-IgG2c-forward CAAgCTCAgAgTACAAAA-gAgCAC; Mus-IgG2c-Reverse CgggAgATggTCTTAgTCgTAA and Mus-IgG2c probe 6FAM-CAggCgAAAAgACTTCCTCTTTCCCA—TMR. Quantitative TaqMan® PCR was performed using Light Cycler FastStart DNA MasterPLUS reaction kits on a LightCycler®480 instrument (Roche Diagnostics) at hybridization temperature of 60 °C.

### 3.11. Tissue collection for Mc detection

Mice were anesthetized with isoflurane, then euthanized in a CO2 chamber. Each mouse was carefully dissected on a clean dissecting board to collect the 12 different organs (Suppl. Tables S4–S6). Cleaning of scissors and forceps was done between each isolated organ within the same individual to avoid cross contaminations. Thymus, spleen and lymph nodes (inguinal, mesenteric, brachial, axillary and cervical) were individually put through a 70 µm sieve with PBS. The cell suspension was centrifuged at 1800 rpm for 10min to obtain a dry pellet of cells for DNA extraction. The other organs (i.e. liver, kidneys, ...) were punctured with a piece of approximately 1 mm³ for DNA extraction.

### 3.12. DNA extractions from tissues and blood cells

Genomic DNA was extracted from pieces of extracted tissues or dry pellet of cells with the EZ1&2 DNA Tissue Kit (Qiagen). Pellets of white blood cells from blood draws were readjusted to a volume of 350 µL with PBS and extracted with the EZ1&2 DNA Blood 350 µL kit, using the EZ1® Advanced XL instrument (Qiagen) according to the manufacturer's instructions.

### 3.13. Quantification of total DNA for Mc research

Total concentration of genomic DNA in each sample tested for Mc was evaluated by amplification of the β actin gene, using a standard reference curve, similarly to previously described method [17]. All re- sults of DNA concentration are expressed in genome equivalent of Mc cells (gEq), with the consensus correspondence of 1 cell (~6 pg): 1 gEq. The primers and the probe were synthesized by TIB MolBiol (Berlin, Germany). Quantitative TaqMan® PCR was performed using Light Cycler FastStart DNA MasterPLUS reaction kits on a LightCycler®480 instrument (Roche Diagnostics).

### 3.14. Quantification of fetal and/or maternal DR4 Mc

HLA-DR4 specific primer and probe sets were synthesized by TIB MolBiol (Berlin, Germany) as previously described [9]. PCR assay sensitivity per reaction-well was of 1 gEq of microchimeric cell in 20, 000 gEq of host cells (0.005 %), thus DNA samples were adjusted to maximum ~20,000 gEq (132 ng) per well, as higher concentrations could reduce the sensitivity, and were tested for Mc in ten replicate wells (for a combined maximal limit-of-detection of 0.0005 %). Results of Mc are given in gEq of donor cells (fetal or maternal) per million of gEq of host cells (gEq/10⁵).

To distinguish DRB1\*04:04<sup>+</sup> maternal Mc from DRB1\*04:01<sup>+</sup> fetal Mc in mice having potentially received both DR4 sources, an allelic HLA-DRB1\*04:04 specific PCR has been designed to not cross react with DRB1\*04:01. Then, samples positive for HLA-DR4 generic PCR were tested by HLA-DRB1\*04:04 allelic PCR. When results were negative for this allelic PCR, samples could be considered as containing only DRB1\*04:01 fetal Mc and not DRB1\*04:04 maternal Mc.

### 3.15. Statistical analysis

Statistical analyses were conducted using GraphPad Prism 9.1.2 software (La Jolla, CA, USA) and the R package. To compare mPAD2 immunized mice and PBS injected mice for their anti-mPAD2 antibody production, or to compare ACPA production from unmated DBA/2 mice versus DBA/2 mice exposed to Mc by PepStarTM peptide microarray technique we used the Fisher exact test. To compare ACPA positivity over time by CCP2 method between unmated DBA/2 vs. DBA/2 exposed to Mc, Kaplan-Meier survival curves were used with ACPA positivity modeled as a final outcome at a "time" event and corresponding log-rank p-values computed for right-censored data. This last analysis allows to statistically evaluate whether the occurrence of the immune response is faster in one group compared to the other. The non-parametric Mann-Whitney test was used to compare quantities of DR4 Mc between ACPA<sup>+</sup> and ACPA<sup>-</sup> females with a DR4 source of Mc. A p-value <0.05 was considered significant.

## 4. Results

### 4.1. Best conditions to obtain 100 % of DBA/2 females positive for DR4 Mc

A mouse having DR4 Mc in at least one of the 13 tissues tested was considered positive for Mc. We thus tested the best conditions in the three different crosses (Fig. 1A, B and 1C) to obtain 100 % of the mice positive for Mc to later test the contribution of Mc to ACPA production.

In crosses allowing exposure to DR4 FMc, we found that one gesta- tion allowed only 40 % of DBA/2 females to be microchimeric in at least one of the tissues tested, while two or three gestations allowed 100 % of females to be positive (See Suppl data and Table S4).

In crosses allowing exposure to DR4 MMc and LMc, we found that DBA/2 mice from a hemizygous H-2<sup>d/b</sup> DR404<sup>+/−</sup> mother were all positive for DR4 Mc at 4–6-month-old while at 1–2-month-old only 25 % were positive (See Suppl data and Table S5).

In crosses allowing exposure to the three sources of DR4 Mc, one gestation with a KO/KI\*04:01 male was sufficient in mice born of hemizygous DR404<sup>+/−</sup> mothers to obtain 100 % of DBA/2 mice positive for DR4 Mc. A second gestation, on the contrary, decreased the per- centage (67 %) of mice positive for DR4 Mc. (See Suppl data and Table S6).

### 4.2. DBA/2 mice with a DR4+ offspring and a DR4+ mother produced more often ACPAs (CCP2 kit) than unmated DBA/2 mice

Prior to analyze ACPA production, we verified that all mPAD2 immunized DBA/2 mice had developed anti-mPAD2 type G
immunoglobulins (IgG) (Suppl. Fig. S2). All the immunized mice produced anti-mPAD2 IgG while none of the DBA/2 control mice injected with PBS (0/8) did (Fisher exact test, $p < 10^{-8}$). Murine PAD2 immunization was efficient after two injections although anti-mPAD2 IgG production was slightly lower in DBA/2 mice than in KO/KI\*04:01 and KO/KI\*04:04 mice positive controls.

DBA/2 females exposed or not to Mc were thus tested for their capacity to produce ACPAs after mPAD2 immunization. Unmated DBA/2 mice were used as negative controls [15]) and KO/KI\*04:01 and KO/KI\*04:04 mice as positive controls [16].

Plasma samples were screened at 60-, 75-, 90- and 105-days post first immunization (Fig. 2) with CCP2 kit, adapted to detect mouse IgG (See material and methods). As expected, none of the ten unmated DBA/2 mice (immunized with mPAD2) produced ACPAs (Fig. 2A) while 100% of the positive control mice KO/KI\*04:01 (Fig. 2B, n = 8) and 75% of the KO/KI\*04:04 mice (Fig. 2C, N = 12) (transgenic mice carrying respectively DRB1\*04:01 or \*04:04 in their genome) did. Among DBA/2 mice exposed to DR4 Mc, ACPAs were produced by 25% of mice with fetal DR4 exposure (N = 12, Figs. s. 2D), 20% of mice with maternal and littermate DR4 exposure (N = 5, Figs. 2E) and 43% of mice with the triple microchimeric DR4 exposure (N = 7, Fig. 2F). The last result was significant when compared to unmated DBA/2 mice (p = 0.027, Fig. 3). Of note, ACPA titers were higher in mice born of DR4<sup>+/-</sup> mothers (Fig. 2E-F) than in mice who had only DR4 offspring (Fig. 2D). Moreover, ACPAs appeared earlier (statistically represented in Fig. 3).

### 4.3. ACPA production is confirmed in DBA/2 mice with the triple DR4 Mc exposure with a very sensitive PepStarTM peptide microarray technique

To test ACPA production with better sensitivity, we used PepStarTM peptide microarrays, a technique previously detailed in Ref. [18] on 20 mouse plasmas (Fig. 4) priorly tested by anti-CPP2 kits.

IgG antibodies specific to citrullinated peptides, or ACPAs, were detected in 1/6 (17%) of unmated mPAD2-immunized mice, 2/5 (40%) of mice with DR4 fetal exposure and 5/5 (100%) of mice with all the sources of DR4 Mc. We thus confirmed that DBA/2 mice born of DR4<sup>+/-</sup> mothers and with DR4<sup>+/-</sup>offspring produced significantly more often ACPAs than unmated mice (Fisher exact test, $p = 0.015$).

### 4.4. The presence of IgG2c isotype in the ACPA response confirmed the microchimeric origin of autoantibodies in IgG2c negative DBA/2 mice

The C57BL/6 strain, from which the Mc cells are derived, secrete IgG2c [19,22] while DBA/2 do not [20]. Although IgG2c is not the dominant isotype in C57BL/6 immune responses (less than 10%), an ACPA production from microchimeric cells should contain IgG2c antibodies.

We tested all the ACPA<sup>pos</sup> DBA/2 mice with DR4 microchimeric exposure for the presence of IgG2c in the ACPA response (CCP2 kit). We could detect the presence of IgG2c in three of the eight (37,5%) microchimeric DBA/2 mice tested (Fig. 5), while none of the 13 unmated DBA/2 mice (3 additional plasma from previous study [15] tested produced IgG2c.

As DBA/2 mice (H-2<sup>d/d</sup> DR404<sup>-/-</sup>) born of hemizygous H-2<sup>d/b</sup> DR404<sup>+/-</sup> mothers has one in two chance of inheriting the maternal IgG2c gene from the C57BL/6 maternal background (Fig. 2B and C), we genotyped all the mice born of such mothers (Fig. 6) and revealed that both mice with highest levels of detected IgG2c protein production (M4Dx3 and M4Dx31, Fig. 5) did not carry IgG2c in their genome. This provides proof-of-concept that the ACPA response in these two mice originates from microchimeric cells.

### 4.5. Quantities of DR4 Mc tended to be higher among ACPA + mice, but Mc was often difficult to detect

DBA/2 mice euthanized 105-120 days post-first mPAD2

immunization were tested for DR4 Mc in blood, lymphoid organs (Table 1) and other organs (Supplementary Table S8). Among the eight ACPA<sup>pos</sup> mice (CCP2 kits), six had DR4 Mc (75%) in at least one tissue of the 13 tested (blood, liver, kidney, etc) compared to 9/16 (56%) of the ACPA<sup>neg</sup> mice. Quantities of DR4 Mc tended to be higher among ACPA<sup>pos</sup> mice (Mann Whitney test, $p = 0.06$). Nevertheless, mice having the highest ACPA titers (i.e. mouse #M4Dx-31, M4Dx3, M4Dx-26) did not have the highest levels of DR4+ Mc. Some even had no detectable Mc at first analysis in the whole organ (i.e. bone marrow M4Dx-31 et M4Dx-32), but low quantities appeared after CD45 cell separation in the CD45<sup>neg</sup> compartment, suggesting the need for more detailed analysis.

## 5. Discussion

A potential role for fetal or maternal cells arising from pregnancy has been suspected to be at the origin of auto-immune disorders [6] without being demonstrated yet. In the present study, we address in an experimental mouse model whether exposure to Mc could contribute to the risk of an autoimmune disease, namely RA, by providing HLA susceptibility alleles and thus contributing to autoimmune response.

We showed two major points. First, DBA/2 mice, previously described [15] and here confirmed as being virtually “unable” to produce RA-specific autoantibodies after PAD immunization, are able to do it when having a mother and/or offspring half transgenic expressing human HLA-DR4<sup>+/-</sup> in a C57BL/6 background [16]. We demonstrated it with two different methods (CCP2 kits and PepStarTM peptide microarrays). Secondly, and importantly, we confirmed the microchimeric origin of autoantibodies using the peculiarity that mice secrete different subclasses of immunoglobulins depending on their strain. As a proof-of-concept, we detected C57BL/6 specific IgG2c isotypes in the autoantibody response of microchimeric DBA/2 mice. One of the limitations of our study is the small number of mice tested *in fine* for our hypothesis despite numerous crossings. This highlights the need for increased sample size in future studies.

Nevertheless, this pilot study conducted here to obtain the maximal acquisition of DR4+ Mc in DBA/2 females was extremely instructive and could explain heterogeneous autoantibody responses between individuals.

Indeed, to test in a mouse model whether pregnancy-derived microchimeric cells could have a role, as hypothesized in Humans, we had to be in the closest conditions to human reproduction where mating is allogeneic. Fetal Mc monitoring in blood and organs, has been well documented *during* mouse pregnancy after congenic or allogeneic mating, and shows lower levels in allogeneic mating compared to syngeneic [23]. But information is scarce when it comes to post-gestational follow-up after allogeneic mating. Similarly maternal Mc has been well documented in mouse fetuses during pregnancy but there are fewer studies in adult mice [24]. We first demonstrated that to obtain persistent FMc in all the mice after allogeneic crosses, at least two successive gestations were required. We also observed that maternal Mc was more often present in 4- to 6-month-old mice versus younger. Moreover, we showed that a situation, where both maternal and fetal sources have a quasi-identical MHC (mother DRB1\*04:04 and offspring DRB1\*04:01), resulted in higher frequency of mice being positive for FMc from the first gestation (80% in that case versus 40% when the mother is not DR4). It is reminiscent to what Kinder et al. called “cross-generational reproductive fitness” [25]. Indeed, the presence of maternal Mc in female offspring enforces their tolerance to the non-inherited maternal antigens (NIMA) when they are sired by males expressing the same MHC alleles than the NIMA [25]. This triggers an MHC compatibility between MMc and FMc in the daughters. Maternal Mc thus enforces tolerance during next generation pregnancy. Interestingly, this effect was not observed at the second gestation, and MMc disappeared. This is in line with a very recent study from Way’s group showing plasticity between MMc and FMc sources, although after pregnancies sired by third party males [26]. This point merits further investigations when repetitive gestations are sired
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Follow up with CCP2 kit of ACPA responses at 60-, 75-, 90- and 105-days post mPAD2 immunization. (A) DBA/2 female mice, without DR4 Mc source (B) C57BL6 KO/KI\*04:01 mice as positive controls for ACPA production; (C) C57BL6 KO/KI\*04:04 mice as positive controls for ACPA production; (D) DBA/2 female mice mated 2 to 3 times with a KOKI404 male and immunized with mPAD2 (with DR404+ fetal Mc contribution), (E) DBA/2 female mice with a hemizygous DR04 mother (DR404+ maternal and littermate Mc contribution); (F) DBA/2 female mice with a hemizygous DR04 mother mated with a KOKI401 male (DR404+ MMc and DR401+FMc). Of note, \#M4D-x3 (red square pointed by a red arrow) has been euthanized at 60 days post immunization but was also positive at 45 days post-immunization with an OD ratio of 53.3. Similarly, the mouse \#M4D-24 pointed by a purple arrow was also positive at 45 days post-immunization, with an OD ratio of 6. The red dotted line defines the threshold to which a sample is considered positive. All samples being at least once positive are represented in color and systematically negative samples in black. Each mouse is represented by a specific symbol and Mouse IDs are indicated on the left side (i.e. \#M4Dx-31 in quadrant E) produces ACPAs from 60 to 105-days post PAD2 immunization).</figcaption></figure>
<figure><img src="image_6.png" /></figure>

(caption on next column)

**Fig. 3.** ACPA positivity modeled as final outcome at a 'time' event between the different group of mice exposed or not to Mc. Kaplan-Meier curves allow to statistically evaluate not only whether there is a difference in response between different groups but also whether the occurrence of the immune response is faster in one group compared to the other. ACPA positivity is modeled as a final outcome at a 'time' event (right-side censoring). P-values are from the long-rank test. (A) comparison of ACPA responses (CCP2 kits) between unmated DBA/2 mice without DR4 Mc exposure (orange curve) and DBA/2 mice with only FMc exposure (green curve), or DBA/2 mice with MMc and LMc exposure (purple curve), or DBA/2 mice with the triple exposure (MMc, LMc and FMc, turquoise curve); (B) comparison of ACPA responses between unmated DBA/2 mice without DR4 Mc exposure (orange curve) and all DBA/2 mice with any Mc exposure (turquoise curve); (C) comparison of ACPA responses between KOKI401 positive controls and KOKI404 positive controls.

with males who express NIMA.

By using anti-CCP2 kits, we observed a higher frequency of ACPA positive mice among those who had both a DR4 mother and DR4 offspring compared to unmated mice; the significant difference was confirmed with a more sensitive PepStarTM peptide microarray technique.

Obviously, a context where ascendants and descendants bring a genetic susceptibility may contribute to a synergistic effect of Mc sources. Moreover, all mice may have received MMc through lactation as they were weaned at 3 weeks and it has been previously demonstrated that lactation can provide additional maternal cells [27,28]. Additionally, we have recently shown that, as a result of multi-embryonic gestation in mice, an exchange of cells between the different fetuses, defined as littermate Mc, can occur and be confounded with the maternal source [17]. In our case, it would be an additional source, possibly increasing the levels of DR4Mc in the group of mice born of hemizygous DR4 mothers. Interestingly enough, we recently showed that fetuses having one or two nearby DR4⁺ littermates in the same uterine horn were almost seven times at higher risk to be DR4 microchimeric in their organs and to have quantitatively more LMc than those without nearby DR4⁺ littermates [17]. This later point highlights interindividual differences according to *in utero* position and may explain why a mouse could have a strong immune response while another from the same litter and same genetics would not (i.e. #M4Dx31 compared to others, Fig. 2E).

Moreover, a phenomenon referred to as compound heterozygosity shows that the shared epitope sequences are most potent in specific combinations (i.e. HLA-DRB1*04:01/*04:04) rather than when they are homozygous (ie: HLA-DRB1*04:01/*04:01 (QKRAA/QKRAA) [29,30]. Therefore, one could wonder whether the higher levels of ACPAs in the group having multiple exposures of different DR4 Mc could come from a compound heterozygosity "reconstituted" in the host [31]. In other word one source of Mc brings one SE⁺ allele and a second source a second SE⁺ allele. A way to test this hypothesis would be to compare our current results with results from mice whose mother and pups were both DRB1*04:01 or whose mother and pups were both DRB1*04:04. But the limitation in the number of animals (3Rs rule) and the heaviness of the crossbreeding and testing experiments alone explain why we have not tested all the conditions. We have also taken the choice to have two slightly different sources of Mc to be able to distinguish them by molecular analyses (Q-PCR), but it would definitely be instructive to make these new crossings.

Noticeably, although 75 % of the ACPA<sup>pos</sup> mice had DR4⁺ Mc versus 56 % of the ACPA<sup>neg</sup> mice, some mice, having among the highest ACPA titers (i.e. mouse #M4Dx-3), were negative for DR4⁺ Mc. A pragmatic response to address this observation is that we have not found DR4Mc in some ACPA<sup>pos</sup> mice because we have not analyzed the host tissues in sufficient detail, which is a limit to the study. Indeed, the equivalent of ~200,000 cells was analyzed per organ but in-depth cell phenotyping would be necessary to isolate Mc cells within lymphoid organs. As a convincing example, in a mouse being negative for DR4Mc in the thymus
<figure><img src="image_7.png" /><figcaption>Fig. 4. Percentage of peptides recognized only under their citrullinated form for each plasma mouse with PepStarTM peptide microarrays. NA: non applicable, X axis: list of mice separated in 6 groups according to their strains, immunizations, and source of DR4 Mc.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. Detection and quantification of the C57BL/6 specific IgG2c subclass in CCP2 specific response of DBA/2 mice with DR4+ Mc source.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** IgG2a and 2c genotyping in DBA/2 mice born of heterozygous C57BL/6KOKIDR404 and DBA/2 (B6D2-DR4<sup>+/-</sup>) mothers. (A) genotyping of DBA/2 control mouse expressing only IgG2a and not 2c; (B) genotyping of heterozygous B6D2-DR4<sup>+/-</sup> mouse used as positive control for both IgG2a and 2c genes; (D–G) genotyping of DBA/2 mice born from a B6D2-DR4<sup>+/-</sup> mother who could have inherited IgG2c from the C57BL/6 background of their mother. Of note only M4Dx33 had inherited IgG2c (E). M4Dx34 (F) presents low level of IgG2c microchimerism as a very low amplification after 40 cycles shows up; (H–N) genotyping of DBA/2 mice born from a B6D2-DR4<sup>+/-</sup> mother and crossed with a C57BL6 KOKI401 male. Mice M4D22 (H), M4D24 (J) and M4D25 (K) have inherited the maternal IgG2c gene.</figcaption></figure>
<caption>**Table 1**</caption>

Evaluation of DR4⁺ Mc in blood and lymphoid organs in DBA/2 mice after various crosses 105 days after the first mPAD2 immunization.

<table><thead><tr><th rowspan="2">Mouse ID</th><th rowspan="2">DR404 mother</th><th rowspan="2">Number of gestations</th><th rowspan="2">CCP2 test</th><th rowspan="2">Peptide arrays<sup>a</sup></th><th rowspan="2">Blood cells (euthanasia)</th><th colspan="4">Post-mortem lymphoid organs</th></tr><tr><th>Thymus</th><th>Bone marrow<sup>b</sup></th><th>Lymph nodes</th><th>Spleen</th></tr></thead><tbody><tr><td>F4D-1</td><td>no</td><td>2</td><td>neg</td><td>nt</td><td>0</td><td>133</td><td>0</td><td>0</td><td>0</td></tr><tr><td>F4D-2</td><td>no</td><td>2</td><td>pos</td><td>nt</td><td>1</td><td>0</td><td>0</td><td>24</td><td>0</td></tr><tr><td>F4D-4</td><td>no</td><td>2</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>9</td><td>0</td></tr><tr><td>F4D-5</td><td>no</td><td>2</td><td>pos</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>979</td><td>4</td></tr><tr><td>F4D-6</td><td>no</td><td>2</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>F4D-10</td><td>no</td><td>2</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>F4D-12</td><td>no</td><td>2</td><td>neg</td><td>nt</td><td>0</td><td>13</td><td>0</td><td>nd</td><td>0</td></tr><tr><td>Mx4</td><td>no</td><td>2</td><td>pos</td><td>neg</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Mx3</td><td>no</td><td>3</td><td>neg</td><td>neg</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Mx6</td><td>no</td><td>3</td><td>neg</td><td>neg</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Nx2</td><td>no</td><td>3</td><td>pos</td><td>pos</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Nx5</td><td>no</td><td>3</td><td>neg</td><td>pos</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx-31</td><td>yes</td><td>0</td><td>pos</td><td>nt</td><td>0</td><td>0</td><td>5<sup>a</sup></td><td>0</td><td>0</td></tr><tr><td>M4Dx-32</td><td>Yes</td><td>0</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>5<sup>a</sup></td><td>0</td><td>0</td></tr><tr><td>M4Dx-33</td><td>yes</td><td>0</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx-34</td><td>yes</td><td>0</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>4</td><td>0</td></tr><tr><td>M4Dx-36</td><td>yes</td><td>0</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx22</td><td>yes</td><td>1</td><td>neg</td><td>pos</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx23</td><td>yes</td><td>1</td><td>neg</td><td>pos</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx24</td><td>yes</td><td>1</td><td>pos</td><td>pos</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx25</td><td>Yes</td><td>1</td><td>neg</td><td>pos</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx26</td><td>Yes</td><td>1</td><td>pos</td><td>pos</td><td>0</td><td>49</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx-1</td><td>Yes</td><td>1</td><td>neg</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>M4Dx-3</td><td>yes</td><td>1</td><td>pos</td><td>nt</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

DBA/2 female mice mated up to three times with transgenic KO/KI*04:04 males (12 mice at the top of the table); or having only a hemizygous H-2d/b DR404⁺/⁻ mother (five mice in the middle of the table) or having both a hemizygous H-2d/b DR404⁺/⁻ mother and mated once with transgenic C57BL/6 KO/KI*04:01 males (seven mice at the bottom of the table) were immunized with mPAD2 and euthanized at 105–120 days post first immunization to quantify DR4⁺ Mc. All results are expressed in gEq/10⁶ Mc cells of the organ/tissue analyzed. Boxes highlighted in blue correspond to positive results of fetal Mc, in brick to positive results of maternal Mc or littermate Mc. a Some samples were not tested by peptide arrays and noted nt, b two bone marrow samples were separated by CD45 sorting, 5 gEq/10⁶ of DR4⁺CD45⁻ cells were found in mice #31 and #32.

(*Mx1, Suppl. Table S4), we further isolated CD90.2⁺ cells, a marker of mouse thymocytes and mature T cells and could find DR4 Mc. Similarly, two mice negative for MMc in their bone marrow samples (#M4Dx-31 and 32) were found slightly positive in the CD45⁻ compartment after CD45 cell separation. Although behind the scope of the current study, a detailed analysis of cell subpopulations of the 312 tissues dissected here would surely have provided more often positive Mc results. An extensive analysis of the lymph nodes would also have been appropriate in a context of immunization and should be taken into account for future studies.

## 6. Conclusion

We could show that non-susceptible DBA/2 mice, virtually “unable” to produce ACPAs in the hapten/carrier model here presented, achieve this when exposed to Mc carrying RA-susceptibility genes. The presence of rare Mc cells in the host’s tissues even below Q-PCR detection threshold may be functionally important. Although this study ultimately only includes a small number of mice after all the various crosses, it provides proof-of-concept that microchimeric cells arising from pregnancy are able to produce detectable rheumatoid arthritis-specific autoantibodies in a mouse model, despite their low presence. Further studies on higher number of mice are needed to decipher the exact role of Mc cells from different origin in ACPA production but ours already points to a role of Mc in the biology of autoimmune diseases, including RA.

## Funding

INSERM, Fondation ARTHRITIS and ARTHRITIS R&D.

## Conflicts of interest

Authors declare that they have no competing interests.

## CRediT authorship contribution statement

**Marie Hemon:** Conceptualization, Data curation, Formal analysis, Methodology. **Mathilde Giassi:** Conceptualization, Data curation, Formal analysis, Methodology. **Yoen Ghaffar:** Data curation, Formal analysis, Methodology. **Marielle Martin:** Data curation. **Jean Roudier:** Writing – review & editing. **Isabelle Auger:** Conceptualization, Data curation, Formal analysis, Methodology, Writing – review & editing. **Nathalie C. Lambert:** Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing.

## Acknowledgements

We would like to thank Sami B. Kanaan, FHCRC, Seattle, WA USA who provided help on statistical analyses and J.Lee Nelson, FHCRC, Seattle, WA USA who provided language help by reading the revised version. We thank our colleagues from Genetic Engineering and Mouse Transgenesis department at service unit CIPHE (Centre d'Immunophénomique, Aix Marseille Université, INSERM, CNRS, Phenomin,
Marseille, France). Particularly Frederic Fiore who performed the generation of the C57BL/6 KOKI mice that greatly assisted our project. We also warmly thank Hervé Luche, Lillia Hadjemand and Djamel Guemboura who provided expertise and advices in the development of pilot immunophenotyping strategies for lymphoid tissues.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jaut.2024.103238.

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