RÉSUMÉ
Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.
Sujet(s)
Affinité des anticorps , Lymphocytes B , Centre germinatif , Anticorps anti-VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Centre germinatif/immunologie , Animaux , Souris , Humains , Lymphocytes B/immunologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/immunologie , Anticorps anti-VIH/immunologie , Affinité des anticorps/immunologie , Anticorps neutralisants/immunologie , Infections à VIH/immunologie , Vaccins contre le SIDA/immunologie , Récepteurs pour l'antigène des lymphocytes B/métabolisme , Récepteurs pour l'antigène des lymphocytes B/immunologie , Techniques de knock-in de gènes , Souris transgéniques , Anticorps neutralisants à large spectre/immunologie , Souris de lignée C57BLRÉSUMÉ
Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.
Sujet(s)
Vaccins contre le SIDA , Anticorps neutralisants à large spectre , Centre germinatif , Anticorps anti-VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Rappel de vaccin , Nanoparticules , Vaccins à ARNm , Animaux , Humains , Souris , Vaccins contre le SIDA/immunologie , Lymphocytes B/immunologie , Anticorps neutralisants à large spectre/immunologie , Réactions croisées , Techniques de knock-in de gènes , Centre germinatif/immunologie , Anticorps anti-VIH/immunologie , Protéine d'enveloppe gp120 du VIH/immunologie , Protéine d'enveloppe gp120 du VIH/composition chimique , Protéine d'enveloppe gp120 du VIH/génétique , Infections à VIH/immunologie , Infections à VIH/prévention et contrôle , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/immunologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Liposomes , Cellules B mémoire/immunologie , Récepteurs pour l'antigène des lymphocytes B/immunologie , Récepteurs pour l'antigène des lymphocytes B/génétique , Hypermutation somatique des gènes des immunoglobulines , Vaccins à ARNm/immunologie , Femelle , Souris de lignée C57BLRÉSUMÉ
Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.
Sujet(s)
Anticorps neutralisants à large spectre , Nanoparticules , ARN messager , Animaux , Souris , Humains , ARN messager/immunologie , ARN messager/génétique , Nanoparticules/composition chimique , Anticorps neutralisants à large spectre/immunologie , Anticorps anti-VIH/immunologie , Lipides/immunologie , Infections à VIH/immunologie , Vaccins contre le SIDA/immunologie , Anticorps neutralisants/immunologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/immunologie , Femelle , Anticorps monoclonaux , LiposomesRÉSUMÉ
Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.
Sujet(s)
Anticorps neutralisants , Anticorps antiviraux , Virus de la grippe A , Vaccins antigrippaux , Infections à Orthomyxoviridae , Vaccination , Animaux , Souris , Humains , Anticorps antiviraux/immunologie , Vaccins antigrippaux/immunologie , Virus de la grippe A/immunologie , Anticorps neutralisants/immunologie , Infections à Orthomyxoviridae/immunologie , Infections à Orthomyxoviridae/prévention et contrôle , Glycoprotéine hémagglutinine du virus influenza/immunologie , Substitution d'acide aminé , Lymphocytes B/immunologie , Grippe humaine/immunologie , Grippe humaine/prévention et contrôle , Anticorps neutralisants à large spectre/immunologieRÉSUMÉ
Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.
Sujet(s)
Anticorps , Production d'anticorps , Lymphocytes B , Usage des codons , Chaines lourdes des immunoglobulines , Inosine , ARN de transfert , Production d'anticorps/génétique , Codon/génétique , Inosine/génétique , Inosine/métabolisme , ARN de transfert/génétique , Anticorps/génétique , Humains , Lymphocytes B/immunologie , Chaines lourdes des immunoglobulines/génétiqueRÉSUMÉ
Monoclonal antibody L9 recognizes the Plasmodium falciparum circumsporozoite protein (PfCSP) and is highly protective following controlled human malaria challenge. To gain insight into its function, we determined cryoelectron microscopy (cryo-EM) structures of L9 in complex with full-length PfCSP and assessed how this recognition influenced protection by wild-type and mutant L9s. Cryo-EM reconstructions at 3.6- and 3.7-Å resolution revealed L9 to recognize PfCSP as an atypical trimer. Each of the three L9s in the trimer directly recognized an Asn-Pro-Asn-Val (NPNV) tetrapeptide on PfCSP and interacted homotypically to facilitate L9-trimer assembly. We analyzed peptides containing different repeat tetrapeptides for binding to wild-type and mutant L9s to delineate epitope and homotypic components of L9 recognition; we found both components necessary for potent malaria protection. Last, we found the 27-residue stretch recognized by L9 to be highly conserved in P. falciparum isolates, suggesting the newly revealed complete L9 epitope to be an attractive vaccine target.
Sujet(s)
Antipaludiques , Vaccins contre le paludisme , Paludisme , Humains , Épitopes , Cryomicroscopie électronique , Plasmodium falciparum , Anticorps antiprotozoaires , Protéines de protozoaire/génétique , Protéines de protozoaire/composition chimiqueRÉSUMÉ
Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.
Sujet(s)
Infections à VIH , Séropositivité VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Vaccins , Souris , Humains , Animaux , Anticorps neutralisants à large spectre , Anticorps neutralisants , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Anticorps anti-VIH , DNA nucleotidylexotransferase , Régions déterminant la complémentarité/génétique , Infections à VIH/prévention et contrôleRÉSUMÉ
Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.
Sujet(s)
Vaccins contre le SIDA , Infections à VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Souris , Humains , Animaux , Anticorps anti-VIH , Anticorps neutralisants à large spectre , Anticorps neutralisants , ARN messager/génétique , Produits du gène env du virus de l'immunodéficience humaineRÉSUMÉ
Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.
Sujet(s)
Lymphocytes B , Centre germinatif , Animaux , Anticorps neutralisants , Anticorps antiviraux , Antigènes , Épitopes , Immunité humorale , SourisRÉSUMÉ
Looking back at the journal's first issue in January 1982 provides an opportunity to reflect on its historical development and to introduce upcoming initiatives.
RÉSUMÉ
Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.
Sujet(s)
Sous-populations de lymphocytes B/immunologie , Épitopes/immunologie , Vaccins contre le paludisme/immunologie , Paludisme/immunologie , Plasmodium falciparum/physiologie , Protéines de protozoaire/immunologie , Vaccins à ADN/immunologie , Transfert adoptif , Animaux , Anticorps antiprotozoaires/métabolisme , Modèles animaux de maladie humaine , Épitopes/génétique , Génie génétique , Humains , Échappement immunitaire , Immunogénicité des vaccins , Souris , Souris SCID , Protéines de protozoaire/génétique , Relation structure-activité , VaccinationRÉSUMÉ
An interview with Facundo D Batista, The EMBO Journal new Editor-in-Chief.
Sujet(s)
Biologie moléculaire/histoire , Allergie et immunologie/histoire , Histoire du 20ème siècle , Histoire du 21ème siècle , Biologie moléculaire/organisation et administration , Périodiques comme sujetRÉSUMÉ
As the journal transitions from fourth to fifth decade, its fourth and fifth Chief Editor discuss its role in the research process.
RÉSUMÉ
T cells form immunological synapses with professional antigen-presenting cells (APCs) resulting in T cell activation and the acquisition of peptide antigen-MHC (pMHC) complexes from the plasma membrane of the APC. They thus become APCs themselves. We investigate the functional outcome of T-T cell antigen presentation by CD4 T cells and find that the antigen-presenting T cells (Tpres) predominantly differentiate into regulatory T cells (Treg), whereas T cells that have been stimulated by Tpres cells predominantly differentiate into Th17 pro-inflammatory cells. Using mice deficient in pMHC uptake by T cells, we show that T-T antigen presentation is important for the development of experimental autoimmune encephalitis and Th17 cell differentiation in vivo. By varying the professional APC:T cell ratio, we can modulate Treg versus Th17 differentiation in vitro and in vivo, suggesting that T-T antigen presentation underlies proinflammatory responses in conditions of antigen scarcity.
Sujet(s)
Présentation d'antigène/immunologie , Antigènes/métabolisme , Polarité de la cellule/immunologie , Cellules Th17/immunologie , Animaux , Antigène CD28/métabolisme , Différenciation cellulaire/immunologie , Membrane cellulaire/métabolisme , Cellules dendritiques/immunologie , Modèles animaux de maladie humaine , Encéphalomyélite auto-immune expérimentale/immunologie , Encéphalomyélite auto-immune expérimentale/anatomopathologie , Régulation de l'expression des gènes , Génome , Antigènes d'histocompatibilité de classe II/immunologie , Souris de lignée C57BL , Lymphocytes T régulateurs/immunologie , Transcription génétique , Trogocytose , Protéines G rho/déficit , Protéines G rho/métabolismeRÉSUMÉ
B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.
Sujet(s)
Lymphocytes B/métabolisme , Systèmes CRISPR-Cas/génétique , Récepteurs pour l'antigène des lymphocytes B/métabolisme , Animaux , Lymphocytes B/immunologie , Anticorps neutralisants à large spectre/immunologie , Systèmes CRISPR-Cas/immunologie , Lignée cellulaire , Techniques de knock-in de gènes/méthodes , Centre germinatif/immunologie , Centre germinatif/métabolisme , Cellules HEK293 , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/immunologie , Humains , Mâle , Souris , Souris de lignée C57BL , Modèles animaux , Récepteurs pour l'antigène des lymphocytes B/immunologieRÉSUMÉ
Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.
Sujet(s)
Vaccins contre le SIDA/génétique , Vaccins contre le SIDA/immunologie , Anticorps neutralisants à large spectre/immunologie , Évolution moléculaire dirigée/méthodes , Anticorps anti-VIH/immunologie , Immunogénicité des vaccins , Produits du gène env du virus de l'immunodéficience humaine/génétique , Produits du gène env du virus de l'immunodéficience humaine/immunologie , Transfert adoptif , Séquence d'acides aminés , Animaux , Lymphocytes B/immunologie , Anticorps neutralisants à large spectre/composition chimique , Régions déterminant la complémentarité/immunologie , Modèles animaux de maladie humaine , Cellules HEK293 , Anticorps anti-VIH/composition chimique , Humains , Souris , Souris knockout , Précurseurs lymphoïdes B/immunologieRÉSUMÉ
During B-cell activation, the dynamic reorganisation of the cytoskeleton is crucial for multiple cellular responses, such as receptor signalling, cell spreading, antigen internalisation, intracellular trafficking, and antigen presentation. However, the role of intermediate filaments (IFs), which represent a major component of the mammalian cytoskeleton, is not well defined. Here, by using multiple super-resolution microscopy techniques, including direct stochastic optical reconstruction microscopy, we show that IFs in B cells undergo drastic reorganisation immediately upon antigen stimulation and that this reorganisation requires actin and microtubules. Although the loss of vimentin in B cells did not impair B-cell development, receptor signalling, and differentiation, vimentin-deficient B cells exhibit altered positioning of antigen-containing and lysosomal associated membrane protein 1 (LAMP1+) compartments, implying that vimentin may play a role in the fine-tuning of intracellular trafficking. Indeed, vimentin-deficient B cells exhibit impaired antigen presentation and delayed antibody responses in vivo. Thus, our study presents a new perspective on the role of IFs in B-cell activation.
RÉSUMÉ
Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30-50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.
Sujet(s)
Lymphocytes B/immunologie , Systèmes CRISPR-Cas , Techniques de knock-in de gènes/méthodes , Chaines lourdes des immunoglobulines , Animaux , Lymphocytes B/cytologie , Humains , Chaines lourdes des immunoglobulines/génétique , Chaines lourdes des immunoglobulines/immunologie , Souris , Souris transgéniquesRÉSUMÉ
Wiskott-Aldrich syndrome protein (WASp) is a main cytoskeletal regulator in B cells. WASp-interacting protein (WIP) binds to and stabilizes WASp but also interacts with actin. Using mice with a mutated actin binding domain of WIP (WIPΔABD), we here investigated the role of WIP binding to actin during B cell activation. We found an altered differentiation of WIPΔABD B cells and diminished antibody affinity maturation after immunization. Mechanistically, WIPΔABD B cells showed impaired B cell receptor (BCR)-induced PI3K signaling and actin reorganization, likely caused by diminished CD81 expression and altered CD19 dynamics on the B cell surface. WIPΔABD B cells displayed reduced in vivo motility, concomitantly with impaired chemotaxis and defective F-actin polarization, HS1 phosphorylation, and polarization of HS1 to F-actin-rich structures after CXCL12 stimulation in vitro. We thus concluded that WIP binding to actin, independent of its binding to WASp, is critical for actin cytoskeleton plasticity in B cells.