Drives Affinity Maturation Research Articles

Somatic hypermutation introduces bystander mutations that prepare SARS-CoV-2 antibodies for emerging variants

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2-neutralizing antibodies (nAbs). However, several potent SARS-CoV-2 antibodies carry no or only a few mutations, leaving the question of how ongoing SHM affects neutralization unclear. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, for some antibodies, including antibodies of the public clonotype VH1-58, neutralization of Wu01 remained unaffected. Although mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical for Omicron BA.1/BA.2 neutralization. We exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2 neutralizer. These findings broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation but also contributes to antibody diversification, thus increasing the chances of neutralizing viral escape variants.

Evolution of antibody immunity following Omicron BA.1 breakthrough infection

Understanding the longitudinal dynamics of antibody immunity following heterologous SAR-CoV-2 breakthrough infection will inform the development of next-generation vaccines. Here, we track SARS-CoV-2 receptor binding domain (RBD)-specific antibody responses up to six months following Omicron BA.1 breakthrough infection in six mRNA-vaccinated individuals. Cross-reactive serum neutralizing antibody and memory B cell (MBC) responses decline by two- to four-fold through the study period. Breakthrough infection elicits minimal de novo Omicron BA.1-specific B cell responses but drives affinity maturation of pre-existing cross-reactive MBCs toward BA.1, which translates into enhanced breadth of activity across other variants. Public clones dominate the neutralizing antibody response at both early and late time points following breakthough infection, and their escape mutation profiles predict newly emergent Omicron sublineages, suggesting that convergent antibody responses continue to shape SARS-CoV-2 evolution. While the study is limited by our relatively small cohort size, these results suggest that heterologous SARS-CoV-2 variant exposure drives the evolution of B cell memory, supporting the continued development of next-generation variant-based vaccines.

Positive selection of IgG+ over IgM+ B cells in the germinal center reaction

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM+ B cells; IgG+ B cells subsequently increased in frequency, dominating GC responses 14-21days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM+ and IgG+ GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM+ GC B cells. Instead, high-affinity IgG+ GC B cells outcompeted high-affinity IgM+ GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.

A Novel Image Analysis Approach Reveals a Role for Complement Receptors 1 and 2 in Follicular Dendritic Cell Organization in Germinal Centers.

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes, via their Fc (FcRs) and complement receptors (CRs) and storing them for long periods in non-degradative vesicles. Presentation of ICs on FDCs to B cells is believed to drive affinity maturation. CR1 and CR2 are expressed on B cells and FDCs. Cr2 knock out (KO) mice, lacking both receptors, have impaired antibody and GC responses. Utilizing a novel ImageJ macro to analyze confocal fluorescence microscopy images of spleen sections, we here investigate how FDCs in wild type (WT) and Cr2 KO mice behave during the first two weeks after immunization with sheep red blood cells (SRBC). Mice were immunized with SRBC i.v. and spleen and serum samples harvested at various time points. As expected, antibody and GC responses in Cr2 KO mice were impaired in comparison to WT mice. Fewer FDCs were identified in Cr2 KO mice, and these exhibited differential localization and organization in comparison to WT mice. WT FDCs were primarily located within GCs at the light zone/dark zone border. FDCs from WT but not Cr2 KO mice were actively dispersed in GCs, i.e. tended to move away from each other, presumably to increase their surface area for B cell interaction. FDCs from Cr2 KO mice were more often found on follicles outside of the GCs and those within the GCs were closer to the periphery in comparison to WT FDCs. Expression of CR1 and CR2, FcγRIIB, and FcµR increased in FDCs from WT mice during the course of immunization. The results suggest that decreased ability to capture ICs by FDCs lacking CR1 and CR2 may not be the only explanation for the impaired GC and antibody responses in Cr2 KO mice. Poor FDC organization in GCs and failure to increase receptor expression after immunization may further contribute to the inefficient immune responses observed.

Exploration of roles of CD4 T cell help in recruitment of rare B cells to germinal centers

Abstract The elicitation of broadly neutralizing antibody (bnAb) responses is a major goal for developing an efficacious HIV vaccine. The HIV Env trimer is a weak immunogen, but it will be a necessary component in a HIV vaccine immunization schedule to drive affinity maturation to bnAbs. There are substantial biological challenges; bnAb precursor B cells are rare in humans, and B cell responses are typically directed to non-neutralizing immunodominant Env epitopes. HIV bnAb development relies heavily on germinal centers (GC) for affinity maturation. The survival of GC B cells is dependent on help from follicular helper CD4 T cells (TFH). Here, we have studied whether the modulation of TFH cells affect B cell immunodominance and competitiveness of rare B cells. We generated Env-specific TCR transgenic mice specific for a I-Ab MHCII epitope we mapped in Env. By utilizing our previously defined VRC01gHL B cell transfer model (Abbott et al., Immunity 2018), combined with co-transfer of CD4 T cells from our new Env-specific TCR transgenic mouse, we examined the competitive fitness of VRC01gHL B cells responding to Env antigens engineered to exhibit varying affinities for VRC01gHL. Increased availability of Env-specific CD4 T cells accelerated the initial expansion of GC VRC01gHL cells in response to a high affinity immunogen. However, improvements in early GC VRC01gHL cells were not observed for low affinity Env trimer immunogens. We are actively exploring whether slow delivery immunization strategies (Cirelli et al., Cell 2019) enhance the ability of Env-specific CD4 T cells to recruit rare VRC01-class B cells to GCs in mice.

GITR shapes humoral immunity by controlling the balance between follicular T helper cells and regulatory T follicular cells

Follicular helper CD4+ T-cells (Tfh) control humoral immunity by driving affinity maturation and isotype-switching of activated B-cells. Tfh localize within B-cell follicles and, upon encounter with cognate antigen, drive B-cell selection in germinal centers (GCs) as GC-Tfh. Tfh functionality is controlled by Foxp3-expressing Tfh, which are known as regulatory T follicular cells (Tfr). Thus far, it remains unclear which factors determine the balance between these functionally opposing follicular T-cell subsets. Here, we demonstrate in human and mouse that Tfh and GC-Tfh, as well as their regulatory counterparts, express glucocorticoid-induced TNF receptor related protein (GITR) on their surface. This costimulatory molecule not only helps to identify follicular T-cell subsets, but also increases the ratio of Tfh vs. Tfr, both within and outside the GC. Correspondingly, GITR triggering increases the number of IL-21 producing CD4+ T-cells, which also produce more IFN-γ and IL-10. The latter are known switch factors for IgG2c and IgG1, respectively, which corresponds to a concomitant increase in IgG2c and IgG1 production upon GITR-mediated costimulation. These results demonstrate that GITR can skew the functional balance between Tfh and Tfr, which offers new therapeutic possibilities in steering humoral immunity.

MiR-29b directly targets activation-induced cytidine deaminase in human B cells and can limit its inappropriate expression in naïve B cells

Class-switch recombination (CSR) is an essential B cell process that alters the isotype of antibody produced by the B cell, tailoring the immune response to the nature of the invading pathogen. CSR requires the activity of the mutagenic enzyme AID (encoded by AICDA) to generate chromosomal lesions within the immunoglobulin genes that initiate the class switching recombination event. These AID-mediated mutations also participate in somatic-hypermutation of the immunoglobulin variable region, driving affinity maturation. As such, AID poses a significant oncogenic threat if it functions outside of the immunoglobulin locus. We found that expression of the microRNA, miR-29b, was repressed in B cells isolated from tonsil tissue, relative to circulating naïve B cells. Further investigation revealed that miR-29b was able to directly initiate the degradation of AID mRNA. Enforced overexpression of miR-29b in human B cells precipitated a reduction in overall AID protein and a corresponding diminution in CSR to IgE. Given miR-29b’s ability to potently target AID, a mutagenic molecule that can initiate chromosomal translocations and “off-target” mutations, we propose that miR-29b acts to silence premature AID expression in naïve B cells, thus reducing the likelihood of inappropriate and potentially dangerous deamination activity.

The Limit of MHC Class II-driven Selection in Germinal Centers

Abstract B cells undergo iterative rounds of proliferation, hypermutation and selection in the germinal centers (GCs) to generate high affinity, protective antibody. It is now thought that GC B cells with higher affinity BCRs acquire more antigen for processing and presenting in association with MHC class II (MHCII) to T follicular helper (TFH) cells than lower affinity competitors. The resulting increase in T-cell help is hypothesized to be the major factor that drives affinity maturation. In this model for GC B cell selection, the density of peptide/MHCII (pMHCII) complexes available on the B-cell membrane is the major determinant of affinity-dependent selection. To quantify the limits of this model for GC selection, we generated short- and long-term chimeric mice with equal numbers of isogenic, mature B cells that differed by their capacity to express MHCII (MHCII+/+ and MHCII+/−). Separately, both B cell types generated virtually identical humoral responses, including affinity maturation, in response to NP-OVA. In competition, MHCII+/+ B cells were preferentially recruited to early GCs consistent with a critical role for pMHCII density in T:B collaboration. However, once established in GCs, no further competitive advantage was observed during the full course (24 days) of the GC response. During this period, both MHCII+/+ and MHCII+/− GC B cells comparably accumulated VH gene segment mutations and exhibited comparable rates of affinity maturation to the NP hapten. These data indicate that B-cell selection determined by MHCII density is more stringent prior to the establishment of GCs than in the GC itself. This observation may be relevant to our recent observation of the persistence of low affinity GC B cells.

The preferred nucleotide contexts of the AID/APOBEC cytidine deaminases have differential effects when mutating retrotransposon and virus sequences compared to host genes

The AID / APOBEC genes are a family of cytidine deaminases that have evolved in vertebrates, and particularly mammals, to mutate RNA and DNA at distinct preferred nucleotide contexts (or “hotspots”) on foreign genomes such as viruses and retrotransposons. These enzymes play a pivotal role in intrinsic immunity defense mechanisms, often deleteriously mutating invading retroviruses or retrotransposons and, in the case of AID, changing antibody sequences to drive affinity maturation. We investigate the strength of various hotspots on their known biological targets by evaluating the potential impact of mutations on the DNA coding sequences of these targets, and compare these results to hypothetical hotspots that did not evolve. We find that the existing AID / APOBEC hotspots have a large impact on retrotransposons and non-mammalian viruses while having a much smaller effect on vital mammalian genes, suggesting co-evolution with AID / APOBECs may have had an impact on the genomes of the viruses we analyzed. We determine that GC content appears to be a significant, but not sole, factor in resistance to deaminase activity. We discuss possible mechanisms AID and APOBEC viral targets have adopted to escape the impacts of deamination activity, including changing the GC content of the genome.

F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse.

Integrin-dependent interactions between T cells and antigen-presenting cells are vital for proper T cell activation, effector function, and memory. Regulation of integrin function occurs via conformational change, which modulates ligand affinity, and receptor clustering, which modulates valency. Here, we show that conformational intermediates of leukocyte functional antigen 1 (LFA-1) form a concentric array at the immunological synapse. Using an inhibitor cocktail to arrest F-actin dynamics, we show that organization of this array depends on F-actin flow and ligand mobility. Furthermore, F-actin flow is critical for maintaining the high affinity conformation of LFA-1, for increasing valency by recruiting LFA-1 to the immunological synapse, and ultimately for promoting intracellular cell adhesion molecule 1 (ICAM-1) binding. Finally, we show that F-actin forces are opposed by immobilized ICAM-1, which triggers LFA-1 activation through a combination of induced fit and tension-based mechanisms. Our data provide direct support for a model in which the T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse.

APOBEC3 enzymes restrict marginal zone B cells.

In general, a long-lasting immune response to viruses is achieved when they are infectious and replication competent. In the mouse, the neutralizing antibody response to Friend murine leukemia virus is contributed by an allelic form of the enzyme Apobec3 (abbreviated A3). This is counterintuitive because A3 directly controls viremia before the onset of adaptive antiviral immune responses. It suggests that A3 also affects the antibody response directly. Here, we studied the relative size of cell populations of the adaptive immune system as a function of A3 activity. We created a transgenic mouse that expresses all seven human A3 enzymes and compared it to WT and mouse A3-deficient mice. A3 enzymes decreased the number of marginal zone B cells, but not the number of follicular B or T cells. When mouse A3 was knocked out, the retroelement hitchhiker-1 and sialyl transferases encoded by genes close to it were overexpressed three and two orders of magnitude, respectively. We suggest that A3 shifts the balance, from the fast antibody response mediated by marginal zone B cells with little affinity maturation, to a more sustained germinal center B-cell response, which drives affinity maturation and, thereby, a better neutralizing response.

Live Adenovirus Type 4 Recombinants Induce Durable Neutralizing Antibody Responses to Influenza H5 with High Levels of Somatic Hypermutation in Humans

OA16.01 Background: Induction of durable antibody responses capable of neutralizing diverse HIV-1 isolates is the most important goal in HIV vaccinology. Broad and potent HIV Env-specific antibodies isolated from humans are characterized by binding to conserved sites on Env and by high levels of hypermutation. Live virus recombinant vectors may provide prolonged exposure to transgene-encoded antigen and may more potently drive affinity maturation than non-replicating vectors. We report results of a small Phase 1 trial of mucosal immunization with live adenovirus type 4 encoding influenza H5 HA (Ad4-H5-Vtn) as a model antigen. We explored its ability to induce durable, broad neutralizing antibody responses through upper respiratory tract application. Methods: Live Ad4-H5Vtn (103-108 viral particles (VP)) was administered by tonsillar swab or by nasal spray in a dose escalation study. Controls received (1010 VP) as enteric-coated capsules. Neutralizing antibodies were measured by a pseudotype HA lentiviral vector system. B cells were isolated using fluorescent HA probes. Immunoglobulin genes were cloned for mutation analysis, and re-expressed to measure neutralization potency and breadth. Results: The vaccine was well tolerated and symptoms were consistent with mild URI. Tonsillar and nasal immunizations were the most immunogenic routes inducing neutralizing antibody responses at day 60 (ID50=809 and 666 respectively) which rose during the first 6 months. B cells that expanded early were specific for both H1 and H5 and were directed to the conserved stalk, with H5 HA-monospecific cells expanding by 6 months. The median level of hypermutation was 3.6% compared to germline at week 4 and 7.4% at weeks 34–52. However, some antibodies contained 13–34% mutation and neutralized group 1 and 2 viruses. Conclusions: A single dose of live recombinant Ad4-H5 can induce highly mutated antibodies, suggesting that mucosal administration is a promising platform for the induction of HIV-specific neutralizing antibodies.

Genetic Assessment of B-cell Responses Following Immunization with HIV Env Protein and Adjuvants or SHIV (AD8) Infection in Nonhuman Primates

OA16.04 Background: An effective preventive vaccine for HIV-1 should generate potent neutralizing antibody responses against the HIV envelope (Env). However, broadly-neutralizing antibodies from HIV+ individuals often have unique characteristics such as extensive somatic hypermutation (SHM), long CDRH3 regions and VH gene restriction. Therefore, we monitored the development of these characteristics in the preclinical setting. Methods: NHP were immunized with HIV Env clade C protein alone, or with a panel of clinical adjuvants or were infected with SHIV(AD8) virus. Env-specific B cells were sorted, barcoded, deep sequenced, and analyzed using a newly curated NHP Ig reference database. In total we analyzed VH gene usage, SHM, and CDRH3 length from over 58,000 unique Ig heavy chains. Results: The adjuvants tested here did not significantly increase germline divergence compared to unadjuvanted protein, nor did they give rise to B cells with long CDRH3 regions. However, animals and individual reads with higher SHM showed evidence of a narrowed VH gene repertoire and were enriched in the IGHV3Q gene. By comparison, antibodies from SHIV infected animals showed an increase in SHM in animals with better neutralization responses. Better neutralization responses further correlated with higher viral loads and viral Env diversity, indicating that chronic antigen stimulation and diversity is likely a driving force for higher SHM and the development of antibody potency and breadth. Finally, we correlated the presence of 3 VH genes: IGHV4A, 4D and 3J with better neutralization, indicating it may be possible to identify a VH gene signature of neutralization. Conclusions: This study is the first comprehensive comparison of antibody genetics using vaccine and infection models in NHP. Our data suggest that future vaccine regimens should be designed to mimic chronic viral infection, utilizing heterologous Env immunogens and prolonged innate and antigen stimulation to drive affinity maturation and neutralization breadth.

Evidence that Shed Thyrotropin Receptor A Subunits Drive Affinity Maturation of Autoantibodies Causing Graves’ Disease

In Graves' disease, thyroid-stimulating antibodies (TSAb) activate the TSH receptor (TSHR) causing hyperthyroidism. Serum polyclonal TSAb are difficult to study because of their extremely low serum levels. Our objective was to determine whether monoclonal TSAb possess characteristics previously reported for polyclonal autoantibodies in Graves' sera. We studied monoclonal TSAb from three laboratories: six generated from mice with induced hyperthyroidism; and one, M22, a human autoantibody obtained from Graves' B cells. All TSAb with one exception were potent activators of TSHR-mediated cAMP generation, with relatively similar half-maximal stimulatory concentrations. Like polyclonal autoantibodies, monoclonal TSAb were largely neutralized by conformationally "active" (but not "inactive") recombinant TSHR A subunits (the N-terminal cleavage product of the TSHR). Chimeric substitutions of TSHR amino acids 25-30 (the extreme N terminus after removal of the 21 residue signal peptide) abrogated the binding and function of all monoclonal TSAb but with one antibody (TSAb4) revealing a nonidentical epitope. Remarkably, these residues are uninvolved in the M22 epitope determined by x-ray analysis. Finally, flow-cytometric dose-response analyses, not previously possible with polyclonal TSAb, revealed that all monoclonal TSAb, human and murine, bound with lower affinity to their in vivo target, the TSH-holoreceptor, than to the isolated TSHR ectodomain. TSAb function does not require antibodies with identical epitopes, and human autoantibody M22 may, therefore, not represent the full epitopic repertoire of polyclonal TSAb in Graves' disease. Most important, we provide strong evidence that the shed ectodomain (primarily the A subunit) is the primary antigen driving affinity maturation of TSAb producing B cells.

Lymphoid Blast Crisis Transformation and Development of Drug- Resistance in Chronic Myeloid Leukemia Are Driven by Aberrant Somatic Hypermutation

Chronic myeloid leukemia (CML) in chronic phase has a disease-free survival of 87% (DFS; 5 years) and long-term treatment with Imatinib is effective. In a large subgroup of patients with CML, however, the disease ultimately progresses into B lymphoid blast crisis (LBC) with only 6% DFS and resistance to Imatinib develops in virtually all cases. In most cases, acquired resistance to Imatinib can be attributed to somatic mutations within the BCR-ABL1 kinase domain. Whereas BCR-ABL1 kinase mutations are rare in chronic phase CML, such mutations are found in >80% of patients with B cell lineage LBC. Likewise, deletions of the ARF and INK4B genes are rare in chronic phase CML but found in ~50% of B cell lineage LBC.In a search for a B cell lineage-specific mutation mechanism responsible for BCR-ABL1 kinase mutations, we tested the hypothesis that aberrant activation of somatic hypermutation may give rise to drug-resistance and progression of chronic phase CML into LBC. Somatic hypermutation drives affinity maturation of immunoglobulins expressed by germinal center B cells and requires the cytidine deaminase AID. Expression of AID depends on PAX5, a transcription factor that determines B cell lineage commitment of hematopoietic progenitor cells. The dependence of AID expression on PAX5 limits somatic hypermutation to the B cell lineage. Consistent with aberrant activation of somatic hypermutation in B cell lineage LBC, we found both PAX5 and AID expression at the mRNA and protein level in B lymphoid but not myeloid subclones from patient-derived blast crisis CML. However, AID protein levels in LBC clones were 5–10-fold lower than in germinal center B cells. To confirm lineage-specific activation of AID-expression in BCR-ABL1 driven leukemia, we isolated bone marrow from Aid-GFP reporter transgenic mice and transformed the bone marrow cells with BCR-ABL1 under either myeloid (IL3, IL6, SCF) or B lymphoid (IL7) culture conditions. The Aid-GFP reporter drives GFP expression under control of upstream and downstream regulatory elements of the Aid locus (Crouch et al., 2007). BCR-ABL1-induced Aid-expression was only observed under B lymphoid culture conditions and was very heterogeneous among the leukemia cell population: Only about 5–10% of CD19+ B lymphoid leukemia clones express Aid-GFP. In these cells, however, Aid mRNA levels are 240-fold higher than in Aid-GFP-negative cells and even 1.5-fold higher than in normal germinal center B cells. Consistent with these findings, we found aberrant somatic hypermutation of the IGHM, BCL6 and MYC loci as well as evidence of ongoing DNA single-strand breaks at the ARF and INK4B loci in B cell lineage LBC but not myeloid CML clones. Ectopic expression of AID in seven otherwise AID-negative CML cell lines cells leads to the acquisition of Imatinib-resistance and sequence analysis of the Imatinib-resistant clones revealed accumulation of mutations within the BCR-ABL1 kinase domain that cause Imatinib-resistance in patients (e.g. L248V, E225K, T315I). Aberrant expression of AID also caused Imatinib-resistance of CML cells in vivo: NOD/SCID mice were injected with CML cells that were either transduced with AID/GFP or GFP alone. Whereas more than the half of the mice injected with GFP+ CML cells were still alive after 170 days, all mice in the AID/GFP+ CML group died within 54 days after injection despite Imatinib-treatment. Forced expression of the B cell-specific transcription factor PAX5 in otherwise PAX5-negative CML cells resulted in a partial B lymphoid lineage conversion similar to LBC. Of note, ectopic expression of PAX5 also resulted in aberrant AID expression, subsequent acquisition of BCR-ABL1 kinase mutations and development of drug-resistance.We conclude that B cell-specific activation of PAX5/AID-induced aberrant somatic hypermutation provides a genetic basis for the strikingly different outcome of myeloid lineage CML as compared to LBC.

Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

High affinity germinal center B cells are actively selected into the plasma cell compartment

A hallmark of T cell–dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this “quality control” over plasma cell differentiation is likely critical for establishing effective humoral immunity.

A Mathematical Model for Germinal Centre Kinetics and Affinity Maturation

We present a mathematical model which reproduces experimental data on the germinal centre (GC) kinetics of the primed primary immune response and on affinity maturation observed during the reaction. We show that antigen masking by antibodies which are produced by emerging plasma cells can drive affinity maturation and provide a feedback mechanism by which the reaction is stable against variations in the initial antigen amount over several orders of magnitude. This provides a possible answer to the long-standing question of the role of antigen reduction in driving affinity maturation. By comparing model predictions with experimental results, we propose that the selection probability of centrocytes and the recycling probability of selected centrocytes are not constant but vary during the GC reaction with respect to time. It is shown that the efficiency of affinity maturation is highest if clones with an affinity for the antigen well above the average affinity in the GC leave the GC for either the memory or plasma cell pool. It is further shown that termination of somatic hypermutation several days before the end of the germinal centre reaction is beneficial for affinity maturation. The impact on affinity maturation of simultaneous initiation of memory cell formation and somatic hypermutation vs. delayed initiation of memory cell formation is discussed.

Autoanti-red cell antibodies synthesized by patients with infectious mononucleosis utilize the VH4-21 gene segment.

A significant proportion of patients with B cell tumors secrete IgM mAb that recognize a carbohydrate autoantigen (li) on the red cell surface. The majority bear an idiotope (Id) that arises from heavy chains encoded by the VH4-21 gene segment, indicating unusual restriction of autoantibody specificity to a single VH gene. Polyclonal anti-li antibodies are also synthesized transiently by normal B cells following certain infections, and we have analyzed the role of the VH4-21 gene in encoding these antibodies. Levels of Id in sera of patients following infection with EBV or Mycoplasma pneumoniae were significantly raised (p < 0.01), and the Id-positive Ig reacted with patients' red cells. Id-positive B cell clones were established from four EBV-infected patients, and 6/6 of the IgM-Id agglutinated red cells. Nucleotide sequence analysis revealed involvement of the VH4-21 gene in all cases, with remarkably little change from the germ-line sequence. However, D segments were heterogeneous, and light chains differed. These results indicate that the same VH gene is used both by polyclonal autoantibodies produced in response to infection, and by B cell tumors. However, the lack of mutations would not appear to give an opportunity for Ag selection to drive affinity maturation of these autoantibodies.