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Paragraph: For proliferation assay, mice were injected i.p. with 50 mg/kg of BrdU (Sigma-Aldrich) and were killed 24 or 72 hours later. Incorporated BrdU was detected intracellularly using anti-BrdU APC-conjugated antibodies according to the manufacturer's recommendations (BD Biosciences). All data acquisitions and analyses were performed with LSRFortessa (Becton Dickinson) interfaced with BD FACSDiva (Becton Dickinson) and FlowJo (Tree Star) software. Subsets of memory B cells were sorted as CD19 + HEL + YFP + IgM + or IgG + and naive cells as CD19 + HEL + YFP -IgM + using a FACSAriaIII flow cytometer. The purity of the sorted populations varied from 90-95%. Sera HEL-specific Ig concentrations were quantified by ELISA. Plates were coated with HEL and saturated with PBS-5% Milk. Dilutions of sera were added. After incubation (2 hours, 37˚C) and washing, HRP-labeled anti-mouse IgM or IgG antibodies were added. After incubation and washing, bound antibodies were revealed with the substrate O-phenylenediamine and H2O2. The reaction was stopped after 10 min. by addition of 10% SDS and the absorbance read at 492nm in a multiscan spectrometer. Ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. The quantification of IgG or IgM secreting cells was assayed by ELISpot technique . Briefly, plates were coated with HEL. After saturating, the cells were distributed into the micro wells in RPMI1640-2%FCS. The plates were incubated for 12 h at 37˚C, 5% CO2 atmosphere. After extensive wash, plates were incubated with goat anti-mouse IgM or anti-IgG labeled with alkaline phosphatase. After washing, the revealing substrate was added (2,3 mM 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices). Sample means were compared using the Student's t test. Sample means were considered significantly different at p < 0.05. During an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory B cells belonging to the same clone. We have devised an experimental system that permits the comparison between naïve and memory B cells expressing the same antigen receptor and allows marking permanently memory B cells. For that purpose we used SW HEL transgenic mice in a Rag2-deficient background holding a single population of monoclonal B cells, all bearing a high-affinity BCR specific for HEL and capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify antigen-experienced B cells the SW HEL . Rag2 -/mice were crossed with mice where AID transcription induces the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . Since in intact Tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-HEL antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal B cell to reconstitute response in immune-deficient hosts and generate antibody memory. Purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into Rag2-deficient mice together with monoclonal OVA-specific CD4 + T helper cells from OTII.Rag2 -/-TCR transgenic mice. The day after, host mice were immunized with OVA-HEL complexes (Fig 1A) . In these conditions, antigenic challenge resulted in B cell activation and the development of significant numbers of CD19 + HEL + AID/YFP + B cells, which were not detected in non-immunized mice or in mice immunized in absence of helper T cells (Fig 1B) . We followed the early kinetics of this response. The number of HEL-specific B cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (Fig 1C left) the B cells expressing AID/YFP being the dominant population (Fig 1C right) . A fraction of the HEL-specific B cells underwent class switch recombination and at day 14 we recovered both IgM + IgG -AID/YFP + and IgM -IgG + AID/YFP + cell populations (Fig 1B) . B cell expansion and phenotypic changes were accompanied by the production of IgM and IgG HEL-specific antibodies ( Fig 1D) . Two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( Fig 1E) . Coherently we found that while upon adoptive transfer all B cells expressed CD95, only after antigenic challenge most YFP + B cells expressed the germinal center specific marker GL7 (Fig 1F) . In conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with B cell activation and expansion, induction of AID expression, class switch recombination, antigen-specific IgM and IgG antibody production and germinal center formation. We studied the evolution of the B cell response. From two weeks onwards the total number of B cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (Fig 2A) . High titers of HEL-specific IgG were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (Fig 2B) . A population of cells secreting HEL-specific Igs was present in the spleen (Fig 2C) , but not in the BM (not shown) even at the late time points. About 60% of the recovered cells exhibited the phenotype of antigen-experienced ("memory") CD19 + HEL + AID/YFP + expressing either IgM or IgG ( Fig  2D and 2E) . We compared the phenotype of the two AID/YFP + IgM + and AID/YFP + IgM -IgG + memory cell populations recovered with that of the naïve B cells (Fig 2F) . We found that antigen-experience and naïve B cells expressed similar levels of CD62L, CD69 and BAFFR (not shown). Antigen-experienced cells presented sustained expression of CD95 and increased levels of PNA, but the vast majority lost expression of the germinal center marker GL7 present at earlier times post-immunization ( Fig 2F compare to Fig 1F) . Compared to naïve B cells, AID/ YFP + cells expressed higher levels of CD80 and MHC class II and down-regulated expression of CXCR5 (Fig 2F) . These findings indicate that the post-germinal center AID/YFP + B cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being CXCR5 low [23] . We have also compared the patterns of gene expression (RNAseq) by naïve, activated (YFPcells of immunized mice) and both populations of YFP + memory cells. The data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of YFP + memory cells (Fig 3) . mRNA was isolated from sort-purified Naïve (CD19 + HEL + YFP -IgM + ) IgM + IgGor IgM -IgG + HEL + CD19 + YFP + memory B cells from spleen of different recipient mice. Total recommended by the manufacturer. The validated libraries were then subjected to DNA sequencing. The analysis is performed using the R software, Bioconductor packages including DESeq2 and the PF2tools package (version 1.2.9) developed at PF2 (Institut Pasteur). Normalization and differential analysis are carried out according to the DESeq2 model and package (version 1.8.1). Fig 3A shows a representative heat map of the different cells populations. Fig  3B shows 

Late in the immune response persistent B cell numbers were kept by active cell division as a significant fraction of the cells were Ki67 + (Fig 2G left) and incorporated BrdU (Fig 2G middle) . The frequency of BrdU + cells was higher among the AID/YFPcells (15%) than in the major AID/YFP + memory population (3%) and similar between the IgM + and IgM -AID/ YFP + populations ( Fig 2G middle and not shown) . Three days after BrdU pulse populations were clear of BrdU + cells (Fig 2G right) attesting their high division rate. In spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (Fig 2H) . The frequency of Caspase3 + cells was higher among the AID/YFP + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. Importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced YFP + cells. It is not yet known whether the number of antigen-experienced memory B cells correlated to the number of naïve B cells or if it is controlled independently of the initial number of antigenspecific B cells present. To approach this question we transferred different numbers of mature naïve B cells from SW HEL .AID/YFP.Rag2 -/donors (ranging from 10 5 to 5.10 6 ) into Rag2-deficient mice together with an excess of CD4 + T helper cells (10 6 ) and immunize the hosts the day after cell transfer with OVA-HEL in optimal non-limiting quantities. To directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. We studied the amplitude of the immune response by measuring the serum titers of HEL-specific IgG antibodies and enumerating the number of HEL-specific B cells recovered. We found that in the presence of excess T cell help, the levels of the HEL-specific IgGs (Fig 4C) , and both the total number of HEL-specific ( Fig 4A) and of memory YFP + B cells recovered (Fig 4B) , did not correlate to the number of antigen specific naïve B cells initially transferred. Memory B cells are defined functionally by their ability to induce secondary IgG antibody responses upon secondary antigenic challenge. We investigated whether the subsets of AID/ YFP + IgM + and AID/YFP + IgM -IgG + antigen-experienced (memory) B cells persisting at late time points could mount secondary IgG responses and persist after secondary transfer. For this purpose we followed two different experimental strategies. In the first, 5x10 4 cells of either IgM + or IgM -IgG + memory B cells, were transferred with an excess helper OTII CD4 + T cells into secondary Rag-deficient hosts that were boosted with OVA-HEL the day after cell transfer. In the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory B cell subsets was about 10-20% of the initial cell input, exceeding naïve B cell recovery (Fig 5A) , supporting the notion that memory B cells may not require specific ligand recognition to survive (2). One cannot exclude, however, that cross-reactivity of the BCR transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of HEL [24] . Following immunization, the secondarily transferred AID/YFP + IgM -IgG + cells responded promptly with the exclusive production of significant levels HEL-specific IgG thus confirming their memory statute (11) . The AID/YFP + IgM + B cells in response to antigenic boost produced only limited amounts of IgM antibodies (Fig 4B) , little IgG antibodies, but did generate GL7 + B cells more efficiently than the IgG + memory B cell population (Fig 5D) . Thus the IgM + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of IgG + effectors (4). With time antibody levels decayed rapidly suggesting that the number of transferred memory B cells declined in the secondary hosts after antigenic boost. Indeed, IgM + and IgG + memory B cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare Fig 5E and 5A) . In similar experimental conditions, naïve B cells following immunization expanded, acquired AID/ YFP expression and their numbers more than doubled the number initially injected (Figs 5F and 2A). These data suggest that a significant fraction of the memory B cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. Besides long-term survival memory B cells must maintain functional activity in the absence of nominal antigen to be fully effective. To test this we used an alternative approach where memory cells were parked in secondary Rag-deficient hosts for 30 days before re-immunization. We found that under these conditions antigenic challenge resulted in the production of HEL-specific IgG antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (Fig 5G) . The aim of this study was to characterize the fate of activated B cells and the generation of memory B cells. To do this, we adoptively transferred monoclonal B cells into immune deficient hosts followed by immunization in presence of T cell help. This strategy resulted in the development of different B cell memory subsets, namely IgM + and IgG + , as described for in situ generated memory cells [4, 6, 14] . These findings indicate that distinct memory B cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single B cell clone. While studying the respective rate of proliferation of both types of memory B cells, we found the same high rate of proliferation for IgM + and IgG + memory B cells. These results contrast with previous published data.

Question: What was used to quantify the amount of IgM secreting cells?
ELISpot technique