key: cord-0282657-zuf1myy0
authors: Boudigou, Marina; Michée-Cospolite, Magalie; Hémon, Patrice; Grasseau, Alexis; Dantec, Christelle Le; Porchet, Emmanuelle; Jamin, Christophe; Devauchelle, Valérie; Mignen, Olivier; Cornec, Divi; Pers, Jacques-Olivier; Pottier, Laëtitia Le; Hillion, Sophie
title: IL-21 and IFN-alpha have both opposite and redundant role on human innate precursors and memory B-cell differentiation
date: 2021-04-01
journal: bioRxiv
DOI: 10.1101/2021.03.31.437810
sha: e8de33f7ea2635b306d87db0838a9a80cd0bd50d
doc_id: 282657
cord_uid: zuf1myy0

Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-α) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD- CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population (referred to as NARB+) characterized by the expression of marginal zone B-cell markers CD45RB and CD1c. Similar to memory B cells, NARB+ cells were in a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals while maintaining their susceptibility to IL-21 activation-induced apoptosis as observed for the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil and gut-associated lymphoid tissues, supported that NARB+ are uncommitted precursors of human marginal zone B cells.

Memory B cells (MBC) and antibody (Ab) secreting cells (ASC) are the key players of humoral immunity resulting in the clearance of pathogens and the establishment of durable immune protection and supporting successful vaccination strategies (1) .

However, the persistence of autoreactive memory B-cell clones could sustain chronic and autoreactive inflammation leading to systemic autoimmune diseases (2) . Some inherent and stable memory functions include long lifespan, high sensitivity to low doses of antigen, quick and robust proliferation, and rapid differentiation into plasma cells highly contrast with those from naive inexperienced B cells (3, 4) . Accumulated data have demonstrated the high degree of heterogeneity of subsets and their progenitors with ambivalent equivalence in humans (5) . Activated B cells that make cognate interactions in secondary lymphoid organs differentiate along either a follicular or extra-follicular (EF) pathways. In the follicular pathway, antigen-activated B cells interact with T cells and form the germinal center (GC), where they undergo somatic hypermutation, selection, and eventually differentiate toward high affinity long-lived plasma cells or IgG + memory B cells. The EF differentiation of B cells is described as very heterogeneous depending on the tissue localization and subsequently on the nature of the availability of T-cell help as described in the lymph node (6) . In the gut or the spleen, this pathway is mostly T-cell independent but relies on signals provided by innate cells (7) . However, the strength of antigen (Ag) recognition appears as a common crucial regulator (8) . The prototype of this pathway is represented by marginal zone B-cell (MZB) differentiation, leading to the production of short-lived plasmablasts (PBs) involved in bacterial protection and the immune regulatory response (9) .

The cytokines IL-21 and IFN-α are major actors involved in the control of B-cell responses. In secondary organs, the production of IL-21 by follicular helper T cells (Tfh) not only initiates affinity maturation in the GCs and isotype switching but also promotes cell proliferation and plasma cell differentiation (10) (11) (12) . IFN proteins are highly produced by plasmacytoid dendritic cells upon viral aggression and are involved in the development of protective immunity mostly against viruses (13) (14) (15) .

Interestingly, IL-21 and IFN-α signaling have shared involvement in the pathogenesis of chronic and autoimmune diseases as well as in uncontrolled exacerbated inflammation (16) (17) (18) . However, the role of IL-21 and IFN-α role in influencing the reactivation of MBCs or initiating the innate B-cell differentiation pathway is not well understood and often analyzed separately, especially in humans (19, 20) . In the present report, we were interested in exploring the role of both cytokines in the initial events that cause activation and plasma cell differentiation from different human mature Bcell progenitors. Thus, we thus used a canonical gating strategy with IgD and CD27 to sort the four main mature B-cell populations. We then analyzed the early response of B cells to IL-21 and IFN-α signaling following BCR and Toll-like receptor (TLR) stimulation. We showed that both signaling pathways induced different B-cell fates, depending on their maturation status. Furthermore, we uncovered a mature B-cell population IgD + CD27 -CD45RB + CD10 -(referred to as NARB + ) harboring highly versatile "multilineage" differentiation potential either able to differentiate into IgM producing PB upon innate signals or adopt pre-GC features in the presence of IL-21.

Phenotypically, NARB + cells share some attributes with IgD + CD27 + MZB cells but retain a unique naive migratory pattern and are sensitive to IL-21 activation-induced apoptosis. This population differed from Ag-inexperienced naive B cells by exhibiting a pre-activated phospho-signaling response to BCR/TLR stimulation. The costimulation with IFN-α but not with IL-6 or IL-10 overtook the IL-21 dependent inhibition of the differentiation pathway. This functional plasticity towards its cytokine environment contrasted with the stable and redundant responses observed in committed MBC subsets. Therefore, this subset could represent an important crossroad in human between the follicular and marginal zone developmental pathways.

Blood samples from healthy donors were collected. Routine plateletpheresis was performed with the Trima Accel device at the French Blood Establishment (EFS).

After donation, tubing of the leukoreduction system chamber (LRSC) was sealed before removing the collection set from the Trima Accel system. The LRSC were kept at 4°C until cell recovery (within 18 hours).

To isolate Peripheral Blood Mononuclear Cells (PBMCs) from Trima Accel devices, residual human blood from the LRSC was harvested and the LRSC was rinsed with phosphate-buffered saline (PBS). PBMCs were isolated by density gradient centrifugation on Lymphocyte Separating medium, Pancoll human (PAN Biotech).

CD19 + B cells were purified from human PBMCs using the REAlease® CD19 Microbead Kit (Miltenyi Biotec) according to the manufacturer's recommendations with purity greater than 98 %.

After isolation, B cells were stained for 30 min at 4°C into the dark with fluorescein isothiocyanate (FITC)-conjugated anti-IgD (IA6-2), phycoerythrin (PE)-conjugated anti-CD10 (ALB1), phycoerythrin linked to cyanin 5.5 (PC5.5)-conjugated anti-CD27 (1A4CD27), allophycocyanin (APC) linked to Alexa Fluor 700 (AAF700)-conjugated CD19 (J3-119) antibodies. CD10-positive B cells, which correspond to transitional subset, were excluded from the cell sorting. Naive (NA, IgD + CD27 -CD10 -), unswitched memory (USM, IgD + CD27 + CD10 -), switched memory (SM, IgD -CD27 + CD10 -) and double-negative B cells (DN, IgD -CD27 -CD10 -) were sorted. For sorting of CD45RB + and CD45RB -NA B cells, FITC-conjugated anti-CD45RB (MEM-55, Immunotools), combined with PE-conjugated anti-CD10, PC5.5-conjugated anti-CD27, APC-conjugated anti-IgD (IA6-2) and AAF700-conjugated anti-CD19 antibodies, was used. Cell sorting was performed using a MoFlow XDP cell sorter (Beckman Coulter). The purity of B-cell subsets was typically greater than 97 %.

For B-cell phenotyping, the following antibodies were used (Supplementary Table 1 ).

For ABCB1 transporter activity analysis, cells were then washed and incubated at 37°C for 30 min with 200 nmol/L MitoTracker Green FM probes (Molecular Probes, Eugene, Ore). Samples were acquired with a Navios cytometer (Beckman Coulter).

Sorted cells were cultured in 96-well plates (Falcon) at 2×10 5 Interleukin-2 (rh IL-2) (20 ng/mL, ImmunoTools) subsequently referred to as primed stimulation. Recombinant human Interferon-alpha 2a (rh IFN-α2a) (10 µg/mL; ImmunoTools), or recombinant human IL-21 (40 ng/mL; Immunotools) were added to the stimulation. For kinetics studies, B cells were stimulated with or without IL-21 for 12 h (0.5 days), 1.5 days, 2.5 days and 3.5 days.

Viability of stimulated B cells were assessed by flow cytometry using FITC-labeled annexin V and propidium iodide (PI) (Biolegend). For apoptosis assays by liveimaging, IncuCyte Ⓡ Annexin V Green Reagent was added directly in the medium at the dilution 1:200 and B cells were imaged every hour for 3.5 days using IncuCyte Ⓡ Live-Cell Analysis System (Sartorius).

For rescue assays, sorted B cells were cultured with rh IL-21 as previously described and with Human CD40-Ligand Multimer Kit (1 µg/mL, Miltenyi Biotec), rh IL-6 (20, 100 or 1000 ng/mL, Immunotools), rh IL-10 (10, 100 or 1000 ng/mL, Immunotools), rh BAFF (50 ng/mL, Immunotools), rh IFN-α2a (10 ng/mL) or with the Pan Caspase Inhibitor Z-VAD-FMK (100 µM, BD Biosciences).

After culture, cultured B cells were harvested, pelleted and the supernatant was collected. Cells were then stained with PE anti-CD11c, PC5.5 anti-CD27, APC-AF700 anti-CD19, APC-AF750 anti-CD38 (Beckman Coulter).Intracellular staining for transcription factors was carried out after cell fixation and permeabilization using the Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer's instructions. Transcription factor expression was analyzed using PE anti-IRF8 (REA516), PE Vio770 anti-IRF4 (REA201), APC anti-BLIMP1/PRDM1 (646702), BV421 anti-T-BET (4B10, Biolegend). Stained B cells were analyzed using a Navios cytometer (Beckman coulter). The phenotype of the cells was analyzed using Kaluza Flow Cytometry Analysis and FlowJo software.

Purified total B cells were first stained with FITC-conjugated anti-CD45RB, PC5.5conjugated anti-CD27, APC-conjugated anti-IgD, AAF700-conjugated anti-CD19 and AAF750-conjugated anti-CD38 antibodies for 30 min at 4°C into dark. After surface staining, cells were stimulated following the primed stimulation with or without rh IL-21 or rh IFN-α2a (as described above) during 5 to 30 min. Unstimulated B cells were used as control. Cells were then fixed in 1.5 % Paraformaldehyde 10 min at room temperature and permeabilized with 100 % ice-cold methanol for 30 min at 4°C. The expression of phosphorylated proteins was measured by flow cytometry using a Navios cytometer with VioBlue (VB)-conjugated anti-pSTAT1 (pY701, REA159), Brilliant Violet 421 (BV421)-conjugated anti-pSTAT3 (pY705, 13A3-1) or BV421conjugated anti-pAKT (pS473, M89-61) (Biolegend) Abs.

Sorted B-cell subsets were labeled with CellTrace Violet reagent (Thermo Fischer Scientific) for 10 min at 37°C prior to the primed stimulation as described above. Bcell proliferation was evaluated by using Navios cytometer and analyzed with FlowJo's Proliferation software.

After 3.5 days of stimulation, the levels of total IgM, IgG, and IgA production by each sorted B-cell subset were evaluated by using the Human IgG/IgM and IgG/IgA FluoroSpot kits (MABTECH), according to the manufacturer's instructions. 15,000 pre-stimulated B cells were incubated by wells during 20 h before revelation. Spots were counted with an Elispot Reader system (Autoimmun Diagnostika GmbH).

For analysis of the intracellular cytokine production, 50 ng/mL Phorbol 12-Myristate 13-Acetate (PMA, Sigma Aldrich), 1 µg/mL ionomycin (Sigma Aldrich) and 10 µg/mL Brefeldin A (Selleckchem) were added for the last 5 hours of culture. Cells were then fixed and permeabilized using Cyto-Fast Fix/perm Buffer Set (Biolegend) according to the manufacturer's instructions and stained with PE anti-IL-6 and PE-Cy7 anti-IL-10 (JES3-9D7) Abs (Biolegend).

Whole blood from healthy controls was analyzed using the Maxpar® Direct™ Immune Profiling Assay™ (Fluidigm) according to manufacturer's instructions on a 

Formalin-fixed paraffin-embedded tissue sections of 4 μ m thickness from gut, tonsil and spleen, were cut onto glass slides. Sections were de-paraffinized with xylene and carried through sequential rehydration from 100% Ethanol to 70% Ethanol before being transferred to Tris-buffered saline (TBS). Heat-induced antigen retrieval was performed in a water bath at 95°C for 30 min in Tris/EDTA buffer (10mM Tris, 1mM EDTA, pH9). Slides were cooled to room temperature (RT) and were subsequently blocked with PBS+3%BSA for 30 min at RT. Each slide was incubated with 100 μ l of the antibody cocktail (Supplementary Table 2 ) overnight at 4°C. Then, slides were washed 3 times with PBS and labeled with 1:500 dilution of Intercalator-Ir (Fluidigm) in TBS for 2 min at RT. Slides were briefly washed with H2O and air dried before imaging mass cytometry (IMC) acquisition. The IMC was purchased from Fluidigm (Fluidigm, Hyperion Imaging System™). Data were acquired on a Hyperion imaging system coupled to a Helios Mass Cytometer (Fludigm), at a laser frequency of of 16-bit single-channel TIFF files were exported from MCD binary files using MCD™ Viewer 1.0 (Fluidigm). Cell-based morphological segmentation was carried out by using supervised pixel classification by Ilastik (64) to identify nuclei, membrane and background, and used CellProfiler (65) to segment the resulting probability maps. Inputs of 16-bit TIFF images with their corresponding segmentation mask were uploaded in histoCAT (66) to open a session data analysis. Dimensionality reduction and unsupervised FlowSOM clustering for 16-bit single images were performed using Cytobank on FCS.files.

Total RNA from ex vivo sorted B cells (NA, SM and DN) and after culture (Primed, IFN-α and IL-21) was extracted using RNA isolation kits (Norgen Biotek) according to the manufacturer's instructions. Libraries were generated by using the Qiaseq RNA library kit (Qiagen) according to manufacturer's instructions. The sequencing analysis was realized by Macrogen society. Fastq reads were aligned using Partek® Flow® Genomic analysis software with STAR aligner and hg38-GENCODE Genes (release 31) as transcript model. Differential expression analysis was performed using the EdgeR package. We treated the PCA loadings onto each of the genes as expression data to run pathway analysis with the PGSEA package. For each pathway, the PAGE algorithm performed one-sample t-test on each gene set in the biological processes branch of Gene Ontology (GO). The adjusted P-values were then used to rank the pathways for each of the first five principal components. The following R code performs PCA and PGSEA analyses: pca.object <-prcomp(t(x)) ; pg = PGSEA (pca,cl=GeneSets(),range=c (15, 2000) , p.value=TRUE, weighted=FALSE). We used the top 500 genes in hierarchical clustering using the heatmap.2 function. The data was centered by subtracting the average expression level for each gene.

From 120 to 500 ng of total RNA were reverse-transcripted with superscript IV 

For in vitro experiments, statistical differences were evaluated using the paired twotailed Wilcoxon signed-rank t-test or repeated measures (RM) one-way ANOVA using Tukey's correction for multiple comparisons when analyzing more than three groups.

All statistical analyses were performed using GraphPad Prism version 8.4.3 and 9

software. Figure 1A) . We analyzed activation and differentiation capacities of the different subsets after activation (anti-BCR, CpG, and IL-2) (23) during a short incubation period (three days) that was inferior to previous culture conditions required for the in vitro class switch recombination (24) . In subsequent experiments, we referred to this stimulation as priming activation. We first followed B-cell activation and differentiation by assessing the expression of CD38 and CD27 in culture to classify resting B cells (CD38 -CD27 -), activated B cells (Act, CD38 + CD27 -), intermediate (Int, CD38 +/-CD27 + ) and plasmablasts (PBs, CD38 high CD27 high ) ( Figure 1A ) adapted from previous studies (18, 25, 26) . As expected, PBs and Int cells were significantly enriched in cultures derived from CD27 + MBCs compared to those from NA and DN cells, which in contrast presented an activated phenotype with enrichment of CD38 + CD27cells.

However, we could detect that 25.9% (± 4.8) of CD38 + CD27 + cells originated from the DN population and more surprisingly 14.2% (± 1.8) from the NA population. To extend these phenotypic observations, we assessed the IgM, IgA, and IgG production by dual-color ELISPOT in activated sorted B-cell subsets ( Figure 1B ). Detection of isotype-specific PB confirmed that the culture model does not allow for the class- Figure 1C ). We robustly detected the induction of IRF4 high IFR8 low cells expressing the PB marker BLIMP1 with no significant difference between the SM, DN, and USM populations regardless of CD27 expression. In agreement with the ELISPOT assays.

NA stimulated cells generated 5.5% (± 1.4) of BLIMP1 + IRF4 + IRF8 low cells that did not acquire a typical CD27 +/high pre-PB/PB phenotype. Moreover, the detection of differentiated cells with functional Ig production derived from NA cells might indicate the existence of a precursor IgD + CD27 -B-cell subset with the ability to rapidly differentiate into IgM + PB.

To investigate the role of IL-21 and IFN-α signaling in mature B-cell differentiation, (28) . Thus, we compared the differentiation response to IL-21 in the IgD + population ( Figure 2B ). In contrast to the effect on IgD + CD27cells, IL-21 had no additional impact on the differentiation of IgD + CD27 + USM cells and did not exhibit a suppressive effect.

IL-21 was initially described to deliver both positive and negative signals in a contextdependent manner in NK cells and B cells (29) and more recently on dendritic cells (30) . To date, several articles have reported a suppressive effect of IL-21 on normal B cells (31) (32) (33) . To understand the opposite role of IL-21 on SM-and NA-derived PB differentiation, we examined the effect of IL-21 on the B-cell apoptosis ( Figure 2C ).

The addition of IL-21 to the initial stimulation significantly decreased annexin V -PIviable NA cells but not viable SM cells. On the contrary, IFN-α significantly promoted NA cell survival, whereas it had no impact on SM cell recovery ( Figure 2C and 2D ).

Multiple rounds of division and differentiation coexist during the reactivation of MBC, balancing in vivo between the generation of either GC or plasma cells (34, 35) .

Furthermore, the functional division of MBCs following reactivation might highly contribute to their regenerative potential. When activated, NA cells remained largely undivided at day 3. In contrast, SM cells proliferated with more than three divisions.

led NA to enter into the cycle ( Figure 2E ). We analyzed the apparition of the IRF4 + IRF8 low PB population in both subsets each day during the culture (Supplementary Figure 2A) . We treated the loadings of the principal components onto each of the genes as expression data to run pathway analysis using the PAGE algorithm on multiple GO pathways and hallmark.MsigDB databases (37) ( Figure 3B ). As anticipated, PC1, representing the culture activation, was strongly associated with the mitotic process, cell cycle, and DNA replication, whereas PC2 correlated with the reticulum process, mRNA translation, and protein production. We then analyzed genes differentially expressed in 

We uncovered the existence of a B-cell population within the IgD + CD27 -NA compartment that rapidly differentiated upon BCR and TLR signaling but had opposite responses between IL-21 and IFN-α. This identity coincided with the down-regulation of some genes involved in MZB differentiation. We thus hypothesized the existence of a MZB-related progenitor cell population displaying multi-potential properties. To explore this idea, we conducted an extensive phenotypic analysis using mass cytometry on circulating peripheral blood (Figure 4) . The CD45RB isoform detected by the Ab clone MEM-55 was reported as an accurate marker to identify not only CD27and CD27 + MBCs (43, 44) but also MZB cell lineage subsets (45, 46) . We thus 

According to these data, we thus hypothesized that this progenitor population might represent the previous B-cell subset among the NA cells that exhibit rapid differentiation upon reactivation. To confirm this hypothesis, we sorted NARB + and NARBcells to evaluate their response in the priming culture. The induction of the IRF4 + IRF8 low BLIMP1 + PBs was only observed in the NARB + cells culture ( Figure   5A and 5B) identifying NARB + as the precursor population of IgM + IgD + CD27 + cells and IgM + PBs. We followed the apoptosis of sorted NARB + and NARBcells by livecell imaging in the primed culture with or without IL-21. NARB + cells conserved functional characteristics of Ag-inexperienced NA cells in response to IL-21 exhibiting rapid activation-cell death with similar kinetics ( Figure 5C ).

We then explored whether these cells could be found in secondary lymphoid organs at steady state. To address this question, we performed imaging mass cytometry on tonsil, spleen, and gut-associated lymphoid tissue sections (Supplementary Figure 4A) .

We visualized and analyzed data using the open-source platform histoCAT, which enables highly multiplexed, quantitative, and detailed analysis of cell phenotypes, microenvironment interactions, and tissue architecture. The workflow of histoCAT consists of overlaying the segmentation masks previously generated by CellProfiler to extract single-cell level information and processing this information into FCS files. We analyzed FCS files analyzed using the t-SNE algorithm that revealed specific cell distribution according to tissue origin ( Figure 5D ). Unsupervised hierarchical clustering using the FlowSOM algorithm was performed to cluster 10 major groups of cells in the different tissues ( Figure 5E ). Although the expression levels of the different markers may significantly vary between them and across the tissues, the heat map view of the FlowSOM clusters identified clusters 1 and 2 were mostly associated with T cells; cluster 3 with macrophages; cluster 4 with neutrophils, and clusters 8, 9, and 10 with B cells. Clusters 5, 6 and 7 had a more ambivalent profile. Finally, Ki67 + proliferative B cells identified the GCs in tonsil and the gut but not in the spleen sections analyzed here (Supplementary Figure 4B) . We confirmed that the clusters overlapped with the main cell populations identified by manual gating. NARB + B cells were detected belonging mostly to cluster 9 ( Figure 5E and Supplementary Figure 4C ). NARB + cells were localized outside the GC mostly in the mantle zone as previously described for IgD + cells. Using neighborhood analysis with histoCAT, NARB + cells were found in very close interaction with CD3 + CD4 + BCL6 + T cells localized at the border of the GCs in the tonsil and in the gut-associated tissues. Moreover, NARB + appeared to be close to Ki67 + proliferative CD3 + T cells that might suggest antigen presentation or activation process. However, the cell interaction map of NARB + differed in the spleen where NARB + were mostly in interaction with innate cells like with CD68 + macrophages and MPO + neutrophils ( Figure 5F and Supplementary Figure   4D ). These data suggested circulating NARB + cells had equivalence in secondary lymphoid tissues that were not restricted to the spleen and might participate in the immune response.

pattern IL-21 signaling via downstream STATs phosphorylations specified the outcome of naive and memory B cells (12, 19) . To shed light on the mechanisms underlying the specific effects of IL-21 on the NARB + population, we compared the early signaling events triggered by IL-21 in combination with the primed stimulation in the different subsets. IL-21 signals through the receptor pair IL-21R/γc, which leads to induction of STAT3 and STAT1 and to a lesser extent the STAT5 and PI3K/AKT pathways. In the priming stimulation, the STAT3 activation showed a similar dynamic of phosphorylation between the different subsets with a peak after 5 min and then a decrease. The STAT1 phosphorylation in USM, SM, and NARB + cells also had a peak of activation after 5 min, however it continued to gradually increase between 30 and 90 min. This increase was not observed for NARBcells ( Figure 6A ). We analyzed the phosphorylation levels of Y705-STAT3 and Y701-STAT1 in the different B-cell subsets after 30 min of activation with or without IL-21 ( Figure 6B ). In the presence of IL-21, NARB + cells presented a dual profile characterized by an important increase of pSTAT3 as observed in Ag-inexperienced NARBcells and a decrease of pSTAT1 as observed in MBC (USM and SM). In addition to STATs activation, the IL-21 proliferative effect was dependent on the PI3K/AKT pathway (49) . We thus examined S473-AKT phosphorylation in the different subsets following activation ( Figure 6C ).

We did not detect a strong additional effect of the addition of IL-21 on the AKT phosphorylation in any of the subsets. However, as observed for STATs phosphorylation, a strong difference existed between the magnitude of the pAKT response following activation between NARBand NARB + cells. Altogether, these data showed that NARB + cells exhibited a specific IL-21-dependent pSTAT1 and pSTAT3 activation pattern. Moreover, NARB + cells have an intracellularphosphorylation signature close to Ag-experienced cells suggesting their previous in vivo activation. Figure 7A ). NARB + produced twice as much IL-6 as NARBat steady state ( Figure   7B ). After in vitro activation, the IL-6 production in NA cells was reduced in the presence of IL-21 whereas IL-10 appeared to be unmodified ( Figure 7C and 7D) . We thus tested whether supplementation of survival factors in the culture might rescue NA cells from IL-21-dependent apoptosis and restore NARB + differentiation. The addition of IL-6 and IL-10 to the IL-21 primed condition at different doses neither improved the viability of NA cells nor the induction of IRF4 + PB in the culture ( Figure 7E ). The CD40 signaling is described as a GC key pro-survival factor and is a hallmark of the provision of T-cell help to B cells. Accordingly, the addition of a CD40L multimer ( Figure 7F and 7G) or BAFF (data not shown) in culture inhibited IL-21-induced apoptosis but did not induce NARB + cells to differentiate, suggesting a subset-intrinsic control of PB differentiation. We then examined whether the biochemical blocking of caspase-induced apoptosis could restore NARB + PB differentiation. We pretreated NA cells with the pan-caspase inhibitor Z-VAD before their activation in the presence of IL-21. As expected, we obtained a full recovery of cell viability in culture, but the PB differentiation was not restored decoupling both mechanisms ( Figure 7F and 7G) .

Lastly, as a test of whether innate signals could overcome IL-21-induced inhibition of NARB + cell differentiation, we analyzed the effect of adding IFN-α to the IL-21 primed condition ( Figure 7H ). Although apoptosis of NA cells was only partially corrected, IFN-α counteracted IL-21 signaling in NARB + cells and re-established PB differentiation.

Taken together, these data support that NARB + cells not only preferentially followed an IFN-α-dependent pathway of differentiation independently of apoptosis but also kept the ability to proliferate in response to IL-21 and T-cell signals.

In this work, we analyzed the effects of IL-21 and IFN-α signals on different subsets of human activated B cells. We used an in vitro B-cell activation model involving BCR and TLR stimulation in presence of IL-2 during a relatively short-time period (referred to as priming cells) (23) . The requirement of permanent BCR engagement with an Ag for MBCs to survive or be reactivated is a major debate (50) (51) (52) , and activation by TLR agonists or bystander T cells help have been suggested to also ensure the longevity of MBC in an Ag-independent process (53) . The priming model (17, 55) . The CD45RB isoform was initially associated with MBC subsets in humans with a high expression on switched CD27 + B cells, PBs, and IgD + IgM + CD27 + USM B cells (45) . More recently, extensive phenotypic and functional analysis of human peripheral B-cell subsets through mass cytometry has refined the CD45RB + CD27population as an early memory B-cell subset based on the level of mutational burden, and their unique metabolic and immune signaling activities, however, with no distinction between isotype status (43) .

In a recent elegant study, the Spencer' group identified CD45RB + T2 cells as progenitors of human marginal zone B cells, suggesting a very close developmental link with the CD45RB + naive population described in this paper (62) . CD45RB + IgD + CD27 -(NARB + ) cells shared different molecular and functional attributes with both naive and memory B cells but with unique IL-21 responsiveness. NARB + cells were able to differentiate upon innate signals toward IgM-producing cells as observed for the IgD + CD27 + (USM) subset. Whereas IL-21 signaling had nearly no impact on the priming activation of USM cells, NARB + cell differentiation is suppressed toward activation-induced apoptosis. The reduction of cell viability in the presence of IL-21 is not restrained to NARB + cells but is a feature of the whole naive compartment. Intracellular STAT and PI3K signaling analysis of the different populations following stimulation demonstrated that the NARB + subset exists in a pre-activated state. This finding challenged the belonging of this subset to the Ag-inexperienced compartment.

Although our data could not trace the trajectory of NARB + differentiation, the phenotypic observations seemed to place this subset in a developmental continuum from naive B cells to MBC. However, another complementary point of view will be to see NARB + cells as circulating innate progenitors of the MZ response. Thus, NARB + cells could be assimilated to a MZP subset, however, the IL-21 typical response observed in vitro suggested that NARB + cells are not fully committed to MZ differentiation and may keep the ability to follow the follicular response in a specific environment. The addition of CD40L signaling to the IL-21 activation cocktail is in favor of this idea, with almost entire cell recovery and entry into the cell cycle. At this stage, we could only speculate on this putative cell pivotal state but its existence in secondary lymphoid organs suggested active participation of the immune response.

Furthermore, our mass cell imaging analyses revealed that NARB + cells seem interact more specifically with innate cells (neutrophils and macrophages) in the spleen, and with pre-Tfh in tonsils and in the gut. These data supported that NARB + cells have specific cell-cell interactions according to their tissue localization that might shape their ultimate function. CD27 dim MBCs were recently described as enriched in infants and might not only represent a reservoir for CD27 high memory effectors but also might enter the GC reaction (56) . The NARB + cells described in this report presented similar functional attributes, suggesting a developmental relationship.

IL-21 production is one of the hallmarks of Tfh identity and is related to the control of the GC B-cell response (39) . However, the IL-21 activity area is not restrained to the follicular response, and BCL6 + PD1 low helper T cells located at the T-B border could contribute to the EF response. There was a defect of EF plasma cells in IL-21R and IL-21 knockout mice. It was further proposed that IL-21 could act at the stage of T-B interaction to accelerate B-cell activation before their differentiation into GC cells or to promote EF PB differentiation (6) . More recently two Tfh effector stages were described according to their spatial positioning in secondary lymphoid organs. EF Tfh cells (PD1 low ) produce high levels of IL-21 and low levels of CXCL13 compared with GC PD1 high CXCR5 high Tfh cells (57) . Therefore, IL-21 availability appears as a key element in the initial activation of B cells (58) . Our data demonstrated that IFN-α could bypass the IL-21 dependent restriction of PB differentiation in NARB + cells.

Early viral responses are mediated by the EF pathway and rely on type I IFN activation (59) Our phenotypic analysis showed that NARB + were a distinct subset from the aNav B cells, as they did not express T-BET and seemed not to be chronically activated (normal expression of CD21 and CD19). Those observations were in favor of a physiological non-pathogenic subset; however, we could not assume any functional relationship between the different EF precursor populations and how they might interact. The control of the EF response and the reactivation of natural innate or chronically activated B cells appear essential in autoimmunity and acute viral infections such as SARS-CoV2 or influenza (61) . The convergent and balanced existence of different EF progenitors could participate in the homeostasis of a such response, providing protective rapid immunity but which could lead to an exacerbated inflammatory response (9) . In this regard, innate MZB cell (USM) differentiation seems impaired in patients with systemic lupus erythematosus and Sjögren's syndrome (63) .Therefore, elucidation of the cellular and molecular actors controlling the initial B-cell activation toward MZB or FO developmental pathway would be promising for better care of systemic autoimmune diseases and hyper-inflammatory syndromes. 

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The authors declare no competing interests. 

The authors would like to acknowledge the Cytometry Core Facility Hyperion (Brest, France) for their technical assistance. We thank Nadège Marec and Pierre Pochard for the assistance in cell sorting, and Patrice Hémon for the HYPERION and CYTOF experiments.

This work has been carried out thanks to the support of the LabEx IGO program (n° ANR-11-LABX-0016-01) funded by the «Investissements d'Avenir» French Government program, managed by the French National Research Agency (ANR)".

The study was performed in accordance with the Declaration of Helsinki and was approved by ethical committees.

The data that support the findings of this study are available from the first and the corresponding authors upon request: sophie.hillion@univ-brest.fr, marina.boudigou@univ-brest.fr BIBLIOGRAPHY