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Paragraph: Protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific "memory" B and T lymphocytes . The generation and selection of T-cell dependent "memory" B cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of AID [1] . Therefore, a long-standing paradigm defined memory B cells as IgM -IgG + isotype switched cells [2] . Different lines of evidence indicate that this is not always the case. In humans, it has been shown that some IgM + B cells bear the phenotype of other memory cells, being CD27 + , and carry frequent point mutations in the V region of the Ig genes, suggesting that they must represent highly selected B cell populations [3] . In mice, populations of CD19 + IgM + able to mount secondary responses have been identified [4] [5] [6] [7] . Overall these findings suggest that the T-cell dependent memory B cell pool comprises distinct subsets of memory B cells with different properties and effector functions [4] [5] [6] . The biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. While initial studies proposed that after transfer memory B cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory B cells were able of extended survival without cell division [11] in the absence of antigen [2] . Long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . The demonstration of the compartmentalization of "antibody memory" into different cellular layers suggested that the separate subsets of memory B cells behave differently. Accordingly, it has been reported that IgG + cells that could rapidly respond upon challenge did not persist long, while IgM + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . Currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. Therefore new therapy strategies may require the adoptive transfer of high-affinity memory B cells, ready to respond and able to persist. The development of these new strategies requires a profound understanding of the mechanisms that regulate memory B cell numbers and ensure long persistence upon adoptive transfer. Moreover, knowledge of the mechanisms that determine the size of the memory B cell pool may be also critical to device new reconstitution strategies. So far, studies comparing populations of naïve and memory B cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory B cells. Indeed in a normal laboratory mouse the population of B cells bearing a "memory IgG + phenotype' represent a small fraction of the total B cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory B cells generated is generally very limited (<10 3 ) [1, 6] . To circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone. We used SW HEL transgenic mice where B cells bear a high-affinity BCR specific for HEL and are capable of class switch recombination and somatic hypermutation (SHM) [17, 18] . To identify "memory B cells" the SW HEL mice were crossed with mice where AID transcription provokes the permanent expression of an YFP reporter in post-germinal center lymphocytes [19] . These mice were in a Rag2-deficient background and therefore contain a pure population of monoclonal HEL-specific B cells. To generate memory cells, purified naïve B cells from the SW HEL .AID/YFP.Rag2 -/mice were transferred into adoptive hosts together with monoclonal OVA-specific CD4 + T cells from OTII.Rag2 -/-TCR transgenic mice. Upon immunization with OVA-HEL complexes, we obtained a significant number of persisting HEL-specific IgM + Ig-G -YFP + and IgM -IgG + YFP + memory B cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory B cell pool is regulated independently. We characterized the functional capacity of these two memory cell types in immune deficient hosts. Mice B6 and B6.Rag2-/- [20] mice were kept at the Centre Des Techniques Avancées (CDTA), Centre National de la Recherche Scientifique (CNRS), Orleans, France; SWHEL.AID/YFP.Rag-/-mice, obtained by crossing SWHEL (18)(a gift of Dr. Robert Brink) and AID/YFP [19] (a gift of Dr. Rafael Casellas) with B6.Rag2-/-mice. OTII.Rag-/-mice were kept in our animal facilities at the Pasteur Institute. Experiments were preformed according to Pasteur Institute Safety Committee in accordance with French and European guidelines and the ethics Committee of Paris 1 (permits 2010-0002, -0003 and -0004). Euthanasia of the mice was performed by cervical dislocation. This specific study was approved by the European Research Council (ERC) committee related to the grant AdG09 249740-QSIS. The general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). The end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. In this case, experiment was stopped and the animals were euthanized. Single-cell suspensions of B cells from spleens and lymph nodes of SW HEL .AID/YFP.Rag -/mice together with CD4 + T cells from spleens and lymph nodes of OTII.Rag -/mice were transferred intravenously into the retro-orbital sinus of B6.Ly5 a IgH a or B6.Rag2 -/recipient mice. Mice received 10 6 HEL + B cells and 10 6 CD4 + T cells unless stated otherwise. Mice were immunized 24H later with 1 mg of Ovalbumin coupled to Hen Egg Lysozyme (OVA-HEL) in 50μg of Alu-S-Gel (Serva) we determined as the optimal dose of Ag (data not shown). Naive cells from SW HEL .AID/YFP.Rag -/mice and memory B cells subsets from immunized B6.Rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. Single-cell suspensions containing 5×10 4 B cells and 10 6 T cells were transferred intravenously into B6.Rag2 -/recipient hosts. The purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of OVA-HEL. Spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, Qdot-655, Brillant Violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-CD19 (6D5), anti-IgM (R6-60.2), anti-IgG1 (X56), anti-CD138 (281-2), anti-Gl7 (Gl7), anti-CD95 (Jo2), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-BAFFR (7H22-E16), anti-CXCR5 (L138D7), anti-IA b (AF6-120.1), anti-CD80 (16-10A1), anti-CD73 (TY-11-8) and anti-PDL2 (TY25) and anti-Ki-67 (mm1) purchased from Becton Dickinson Pharmingen, Biolegend, Invitrogen and eBioscience. Cells were also stained with HEL (Sigma) coupled with AF594 using Alexa Fluor1 594 Protein Labeling Kit from Life technologies. Before staining, cells were treated with Fc-Block (CD16/CD32, Becton Dickinson Pharmingen). Dead cells were excluded during analysis according to their light-scattering characteristics. For intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. Then, cells were fixed and permeabilized according the manufacturer's recommendations (BD Bisciences). 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) .

Question: What is required to establish a secondary immune response to a viral infection?
the generation of antigen-specific "memory" B and T lymphocytes