Memory B-cell Clones Research Articles

Adoptive transfer of donor B lymphocytes: a phase 1/2a study for patients after allogeneic stem cell transplantation

AbstractImmune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is slow and patients carry a high and prolonged risk of opportunistic infections. We hypothesized that the adoptive transfer of donor B cells can foster after HSCT immuno-reconstitution. Here, we report, to our knowledge, the results of a first-in-human phase 1/2a study aimed to evaluate the feasibility and safety of adoptively transferred donor B cells and to test their activity upon recall vaccination. Good manufactoring practice (GMP) B-cell products were generated from donor apheresis products using 2-step magnetic cell separation. Fifteen patients who had undergone allo-HSCT were enrolled and treated after taper of immunosuppression (median, day +148; range, 130-160). Patients received 4 different doses of B cells (0.5 × 106 to 4.0 × 106 B cells per kg body weight). To test the activity of infused donor memory B cells in vivo, patients were vaccinated with a pentavalent vaccine 7 days after B-cell transfer. We observed the mobilization of plasmablasts and an increase in serum titers against vaccine antigens, with a stronger response in patients receiving higher B-cell numbers. Analysis of immunoglobulin VH-sequences by next-generation sequencing revealed that plasmablasts responding to vaccination originated from memory B-cell clones from the donor. Donor B-cell transfer was safe, as no Epstein-Barr virus (EBV) reactivation was observed, and only low-grade graft-versus-host disease (GVHD) occurred in 4 out of 15 patients. This pilot trial may pave the way for further studies exploring the adoptive transfer of memory B cells to reduce the frequency of infections after allo-HSCT. This trial was registered at ClinicalTrial.gov as #NCT02007811.

Influenza vaccination-induced H3 stalk-reactive memory B-cell clone expansion

Current vaccine formulations elicit a recall immune response against viruses by targeting epitopes on the globular head of hemagglutinin (HA), and stalk-reactive antibodies are rarely found. However, stalk-specific memory B-cell expansion after influenza vaccination is poorly understood. In this study, B cells were isolated from individuals immunized with seasonal tetravalent influenza vaccines at days 0 and 28 for H7N9 stimulation in vitro. Plasma and supernatants were collected for the analysis of anti-HA IgG using ELISA and a Luminex assay. Memory B cells were positively enriched, and total RNA was extracted for B cell receptor (BCR) H-CDR3 sequencing. All subjects displayed increased anti-H3 antibody secretion after vaccination, whereas no increase in cH5/3-reactive IgG levels was detected. The number of shared memory B-cell clones among individuals dropped dramatically from 593 to 37. Four out of 5 subjects displayed enhanced frequencies of the VH3-23 and VH3-30 genes, and one exhibited an increase in the frequency of VH1-18, which are associated with the stalk of HA. An increase in H3 stalk-specific antibodies produced by B cells stimulated with H7N9 viruses was detected after vaccination. These results demonstrated that H3 stalk-specific memory B cells can expand and secrete antibodies that bind to the stalk in vitro, although no increase in serum H3 stalk-reactive antibodies was found after vaccination, indicating potential for developing a universal vaccine strategy.

A First Step Toward a Cross-tissue Atlas of Immune Cells in Humans.

A First Step Toward a Cross-tissue Atlas of Immune Cells in Humans.

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

BackgroundSeveral studies have demonstrated neutralizing antibodies to be highly effective against alphavirus infection in animal models, both prophylactically and remedially. In most studies, neutralizing antibodies have been evaluated for their ability to block viral entry in vitro but recent evidence suggests that antibody inhibition through other mechanisms, including viral budding/release, significantly contributes to viral control in vivo for a number of alphaviruses.ResultsWe describe a BSL-2, cell-based, high-throughput screening system that specifically screens for inhibitors of alphavirus egress using chikungunya virus (CHIKV) and Mayaro virus (MAYV) novel replication competent nano-luciferase (nLuc) reporter viruses. Screening of both polyclonal sera and memory B-cell clones from CHIKV immune individuals using the optimized assay detected several antibodies that display potent anti-budding activity.ConclusionsWe describe an “anti-budding assay” to specifically screen for inhibitors of viral egress using novel CHIKV and MAYV nLuc reporter viruses. This BSL-2 safe, high-throughput system can be utilized to explore neutralizing “anti-budding” antibodies to yield potent candidates for CHIKV and MAYV therapeutics and prophylaxis.

Alemtuzumab-induced immune phenotype and repertoire changes: implications for secondary autoimmunity.

Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing–remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points.Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease.In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.

Adoptive Transfer of Purified Donor-B-Lymphocytes after Allogeneic Stem Cell Transplantation: Results from a Phase I/IIa Clinical Trial

Patients after allogeneic stem cell transplantation (alloSCT) show a long lasting immune deficiency involving both T- and B-lymphocytes. Antibody responses to vaccination, as a measure of functional B-cell immunity, are insufficient in the first months after alloSCT and usually recover within 1-2 years. To improve B-cell immune reconstitution after alloSCT, we developed a novel concept of adoptive transfer of memory B-cells from the original stem cell donor (Klenovsek et al., Blood 110:3472-79, 2007). To this end, we first established the technology for the manufactoring of a GMP-qualified B-cell product and initiated a first-in-man phase I/IIa clinical trial in patients after alloSCT to evaluate safety and tolerability of adoptively transferred donor B-cells.B-cells were manufactured under GMP conditions from 16 unstimulated leukapheresis products derived from the original stem cell donor. The separation protocol consists of two subsequent separation steps on the CliniMACS® System (Miltenyi Biotec) including depletion of CD3+ T-cells and subsequent positive separation of CD19+ B-cells by magnetic beads. In all of the 16 cell products high purities of CD20+ B-cells (average 96%, range 85-99%) with only low frequencies of contaminating T-cells (average 0.10 %, range 0.01-0.82%) were achieved. After manufacturing, B-cell products were cryopreserved for single administration to 4 groups of patients with escalating doses. To prevent GvH reactions the absolute number of CD3+ T lymphocytes in the B-cell product had to be below 4 x 104 CD3+ cells/kg BW.The clinical trial was designed as an open label dose escalating study using the traditional 3+3 design with 4 dose levels of 0.5-1-2-4x106 B-cells/kg BW. Patients were enrolled on day +140 ± 20 after alloSCT after tapering of immunosuppression. Patients with acute GVHD grade III/IV, chronic GVHD with intermediate to high risk, EBV reactivation (>10,000 EBV copies/ml) or after therapy with B-cell antibodies (i.e. rituximab) were not eligible. Up to now, 13 patients after alloSCT received donor B-cells in 4 different B-cell dosages (average day +147, range from day +130 to d +160). Three patients received 0.5x106 B-cells per kg BW, 3 patients 1.0x106 B-cells/kg BW, 5 patients 2.0x106 B-cells per kg BW, and 2 patients 4.0x106 B-cells/kg BW, respectively. The median number of infused T cells was 0.06x104 T-cells per kg BW (range 0.01-0.13 x104/kg BW). Regarding primary safety data, the B-cell transfer was well tolerated without any acute adverse reactions. Importantly, neither significant EBV-viremia nor acute or chronic GvHD reactions were observed during the observation period of 4 months after the transfer of B-cells.In addition to the analysis of the primary safety endpoints of the trial, the activity of infused donor memory B-cells in vivo were tested. A characteristic memory B-cell response is defined as a mobilization of plasmablasts (PBs) 7 days after booster vaccination into the peripheral blood (Odendahl et al., Blood 105: 1614-21, 2005). Preliminary results obtained from patients that received the donor derived B-cells demonstrated significant mobilization of PBs in some of the patients after re-vaccination with a pentavalent vaccine (Pentavac®), suggestive for a memory B-cell response. A detailed analysis of the vaccination data from the study patients in comparison to a control group of transplanted patients without B-cell transfer after routine re-vaccination will be presented. Furthermore, the analysis of immunoglobulin VH-sequences by next-generation-sequencing in memory B-cell populations of two individual patients after B-cell transfer revealed that at least 50% of all memory B-cell clones identified in the recipient are unambiguously contained within the memory B-cell population of the infused B-cell product.In summary, our data show that the manufacturing of a B-cell product from the original stem cell donor under GMP conditions without significant T-cell contamination is feasible. The transfer of donor-derived B-cells into patients after alloSCT is safe with regard to EBV reactivation and induction of acute or chronic GvHD. Future clinical trials will evaluate whether the adoptive transfer of memory B-cells from the donor can significantly reduce the frequency of infections after alloSCT.(Supported by the Deutsche Forschungsgemeinschaft through SFB 643 "Strategies of cellular immune intervention") DisclosuresNo relevant conflicts of interest to declare.

Human memory B cells.

A key feature of the adaptive immune system is the generation of memory B and T cells and long-lived plasma cells, providing protective immunity against recurring infectious agents. Memory B cells are generated in germinal center (GC) reactions in the course of T cell-dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation and expression of somatically mutated and affinity matured immunoglobulin (Ig) genes. Approximately 40% of human B cells in adults are memory B cells, and several subsets were identified. Besides IgG+ and IgA+ memory B cells, ∼50% of peripheral blood memory B cells express IgM with or without IgD. Further smaller subpopulations have additionally been described. These various subsets share typical memory B cell features, but likely also fulfill distinct functions. IgM memory B cells appear to have the propensity for refined adaptation upon restimulation in additional GC reactions, whereas reactivated IgG B cells rather differentiate directly into plasma cells. The human memory B-cell pool is characterized by (sometimes amazingly large) clonal expansions, often showing extensive intraclonal IgV gene diversity. Moreover, memory B-cell clones are frequently composed of members of various subsets, showing that from a single GC B-cell clone a variety of memory B cells with distinct functions is generated. Thus, the human memory B-cell compartment is highly diverse and flexible. Several B-cell malignancies display features suggesting a derivation from memory B cells. This includes a subset of chronic lymphocytic leukemia, hairy cell leukemia and marginal zone lymphomas. The exposure of memory B cells to oncogenic events during their generation in the GC, the longevity of these B cells and the ease to activate them may be key determinants for their malignant transformation.

Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM(+) and IgG(+) memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM(+)(IgD(+)) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells--including IgM(+)IgD(+)CD27(+) B cells--are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM(+)(IgD(+)) and class-switched memory B cells.

Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes

The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein-Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases.

Iterative Germinal Center Re-Entries of Memory B-Cells with t(14;18) Translocation and Early Steps of Follicular Lymphoma Progression

AbstractAbstract 150The recent demonstration that memory B-cells can re-enter germinal centers (GCs) and participate to new rounds of GC reactions has opened the possibility that multi-hit B-cell lymphomagenesis could be a much more dynamic process than initially anticipated, gradually progressing throughout the successive passages of memory B-cells in GCs during a lifetime of successive immunological challenges. Here, we provide evidence for this scenario in follicular lymphoma (FL), a GC derived B-cell malignancy initiated in the bone marrow by the hallmark t(14;18) BCL2/IGH translocation. To address this issue, we engineered an original sporadic BCL2tracer mouse model mimicking the rare occurrence of t(14;18) translocation in humans through V(D)J recombination errors (1 in a million B-cells) allowing to track the resultant BCL2-expressing clones; and underwent a molecular/immunofluorescent tracking of t(14;18)+ clones vs. normal memory B-cells in paired lymphoid tissue samples (spleen, lymph nodes, bone marrow) from healthy individuals.We first show that contrary to the current dogma, ectopic BCL2 expression is not sufficient to provoke the FL’s characteristic differentiation arrest of activated B-cells as GC B-cells, thereby suggesting that differentiated BCL2+ memory B-cells must return to the GC to acquire additional oncogenic hits and “fix” in situ growth. Strikingly, we further find that in a small fraction of “healthy” humans, such differentiation arrest already operated, and that a clonally expanded population of t(14;18)+ cells with FL-like features have widely disseminated in blood and in multiple lymphoid organs (spleen, lymph nodes, bone marrow), with unprecedentedly reported frequencies (from 1/million to 1/500 cells in some individuals), shaping the systemic disease presentation observed in FL patients. Using molecular/immunofluorescent backtracking of such clones in various paired and remote lymphoid organs, we further demonstrate that t(14;18)+ clones systematically display an extensive history of AID (activation-induced cytidine deaminase)-mediated events compatible with iterative rounds of GC co-opting, in sharp contrast to single memory B-cell clones from the same individuals. We thus show that BCL2-expressing memory B-cells require multiple GC transits to acquire the distinctive FL-like maturation arrest as centrocyte/centroblasts and to progress to advanced FL precursor stages. Altogether, our data argue for a model of lymphomagenesis, in which progression from precursor stages to FL occurs asymptomatically over an extended period of time by subverting the dynamic and plastic attributes of memory B-cells. This understanding of the pre-clinical phases driving FL development in asymptomatic patients should help rationalize prospective approaches designed to identify biomarkers of risk, and innovative therapeutic targets present in early, potentially more curable phases of the disease. Disclosures:No relevant conflicts of interest to declare.

Inhibitory Spleen-Derived Anti-ADAMTS13 Antibodies Are Characterized by a Limited Number of Variable Heavy Chain CDR3 Signatures in Patients with Relapsing Acquired Thrombotic Thromobocytopenic Purpura

Abstract 194 Background.Acquired Thrombotic thromobcytopenic purpura (TTP) is the result of autoantibodies neutralizing and-or accelerating clearance of ADAMTS13. We have previously isolated mononuclear cells of two spleens donated to our laboratory by patients (A and B) splenectomized for frequently relapsing TTP to generate human monoclonal anti-ADAMTS13 antibodies. Preliminary characterization of the patient’s entire IgG4 repertoire using phage display technology and Epstein Barr virus (EBV) transformation of switched memory B cells (CD19+, CD27+, IgG+) showed that the memory repertoire of Rituximab-resistant (B) and non-resistant (A) anti-ADAMTS13 IgG used the same restricted VH germline genes, namely VH1-3 (55%), VH1-69 (17%), VH3-30 (7%) and 4–28 (21%). In contrast, peripheral blood-derived IgG1 anti-ADAMTS13 antibody repertoire from 2 acquired TTP patients, inhibiting ADAMTS13 activity by maximal 15–40% used VH1-69 germline gene segment only (Luken et al, JTH 2006 and Pos et al, JTH 2009). To evaluate if the generated spleen-derived monoclonal anti-ADAMTS13 antibodies are of pathological relevance we started with their functional characterization. Methods.Both patients (A and B) were suffering from acute TTP with high titers of anti-ADAMTS13-Ab and therefore needed to be splenectomized because of frequent relapses, which occurred even after several courses of Rituximab (B). Both patients have remained in remission since splenectomy. Previous screening of the IgGk-IgGλ IgG4-Fab phage-display libraries and EBV-transformed clones on recombinant ADAMTS13 by ELISA revealed high ADAMTS13 specificity for 16/34 and 13/234 antibodies, respectively. Functional characterization of the phage-display clones included (a) determination of their inhbitiory capacity by functional FRETS inhibitor assay, (b) assessment of their affinity performed by either fluid-phase competition ELISA and/or plasma surface resonance (Biacore), and (c) epitope mapping by dot blot analysis testing their recognition of recombinant full-length wildtype ADAMTS13 and a truncated ADAMTS13 variant. Results.Detailed sequence analysis of all human monoclonal anti-ADAMTS13 analyzed showed seven unique CDR3 signatures in the variable heavy region of all clones generated using both techniques (phage display and EBV-transformation), three of which were seen in both patients. These CDR3 signatures are different from signatures found in peripheral blood-derived antibodies (Luken et al, JTH 2006 and Pos et al, Blood 2010). Strikingly, the anti-ADAMTS13 antibodies of EBV-transformed memory B cells used λ light chain exclusively, whereas the phage-display-derived clones showed usage of either κ- (A: 6/11 and B: 3/5) or λ-light chain (A: 5/11 and B: 2/5). Anti-ADAMTS13 IgG in supernatants of EBV-immortalized switched memory B-cell clones were mainly of subclass IgG4 (A: 60%; B: 25%) and IgG1 (A: 40%; B: 75%) thus IgG1 being the prevailing subclass in patient B, who relapsed despite several courses of Rituximab. Functional characterization of the anti-ADAMTS13 IgG4 antibodies revealed that 15/16 phage display clones are strongly inhibitory, showing 0–100% (A) and 60–100% (B) inhibition at 200 nM and even 0–92% (A) and 0–16% (B) inhibition at 20 nM. This represents a 5–6 times increase of inhibitory capacity when compared to previouse findingy by others, in the range of 2×10−7-7×10−10 M (A) and 1×10−9-6×10−10M (B) as measured by Biacore or/and competition ELISA. Epitope mapping revealed that 3/16 clones recognized a novel epitope at the C-terminus of ADAMTS13, in contrast to the previously reported N-terminally located spacer domain. Conclusions.The identification of several spleen-derived anti-ADAMTS13 clones displaying highly similar CDR3 variable heavy chains, in 2 unrelated acquired TTP patients is striking and novel. Our data points at the discovery of unique CDR3 signatures that characterize the pathological relevant, inhibitory antibodies paving thus the road to define their triggering ADAMTS13 epitope(s), while providing insight into the mode of success of splenectomy in relapsing TTP patients. Further functional characterization of the anti-ADAMTS13 EBV-derived clones and confirmation of our data by analysis of the immunological memory from two additional spleens are underway. Disclosures:No relevant conflicts of interest to declare.

Author reply

Recently we demonstrated that, after chemotherapy, pediatric patients frequently lose the protective humoral immunity induced by vaccination, with the degree of loss varying according to the type of vaccination (52% for hepatitis B, 25% for measles, 21% for mumps, 18% for rubella, 13% for tetanus, and 8% for polio).1 It is interesting to note that the administration of a booster vaccination to 51 patients enabled all but 3 patients to recover protective antibody titers. In their study, Fioredda et al. did not find a comparable percentage of loss of hepatitis B antibody in a smaller study conducted at two other Italian pediatric centers.2 In view of this, they suggest continuing the vaccination schedule on the basis of the patient's age and do not advocate any booster immunizations. In contrast to our study, the effect of chemotherapy on the loss of protective antibody titers is not clear from these data because of the lack of any assessment of the patient's serologic status before chemotherapy. Differences in patient age at the time of diagnosis, type of malignancy, chemotherapy protocol, and the time to assessment of the antibody levels after the completion of chemotherapy may explain in part the differences between the two studies. Regardless, a major impairment of humoral immunity against hepatitis B has been reported previously in other immunocompromised patients such as those who are positive for the human immunodeficiency virus and patients undergoing hemodialysis.3, 4 Conversely, Laws et al. state that higher losses of protective antibody titers have been observed in children after chemotherapy (20–70% of children for diphtheria and in up to 72% of children for polio). These authors consider that revaccination is the best tool with which to reconstitute protective antibody titers, although their reported response rate was as low as 30% after the first booster and only 60% after a second booster vaccination for diphtheria.5 These data suggest that standard nonmyeloablative chemotherapy can definitely delete specific memory B-cell clones obtained by vaccination and that no spontaneous recovery is expected. This point requires further confirmation but, if true, the lack of epidemiologic data concerning the incidence of vaccine-preventable diseases among children after chemotherapy may reduce the strength of the case for revaccination. In other words, we do not know whether these children are really at risk of developing such diseases or if the immunity of the rest of population will protect them, at least for diseases such as diphtheria and polio.6 The different opinions expressed by several authors clearly reflect the diversity of data regarding vaccine-specific immune recovery after standard chemotherapy. This point is crucial to deciding which strategy of (re-)immunization after chemotherapy (if any) is needed and further prospective studies concerning this important issue are advocated. Simone Cesaro M.D.*, Ruggiero D'Elia M.D. , * Clinic of Pediatric Hematology Oncology, Department of Pediatrics, University of Padova, Padova, Italy, Service of Infectious Disease in, the Pediatric Immunocompromised Host, Department of Pediatrics, University of Padova, Padova, Italy

Memory B-cell clones and the diversity of their members

Memory B-cell clones develop from virgin B cells that take up processed antigen, make cognate interaction with primed T cells and then grow in germinal centres. Within the germinal centre the proliferating B cells undergo Ig variable-region mutation and are subsequently selected on their ability to bind antigen held on follicular dendritic cells and then to make cognate interaction with germinal centre T cells. The selected cells emerge as memory B cells or plasmablasts. Although many of the memory B cells and most of the plasma cells emerging from follicles have undergone Ig class switch recombination a substantial minority of the memory B cells have not switched. These non-switched memory cells can be induced to switch on re-exposure to antigen. Affinity maturation following a single immunization ceases as germinal centres wane some 3–4 weeks after immunization — memory cells and antibody production, on the other hand, persist for months and even years.

The cellular origins of memory B cells

Recent evidence indicates that memory B cells may originate from a precursor cell subset that is distinct from AFC precursors. Most convincing is the finding that fractionation of naive peripheral B-cell populations on the basis of surface heat stable antigen (HSA) expression yields two populations; one greatly enriched for progenitors of memory B cells (HSAlo), and the other enriched for AFC precursors (HSAint-hi). Antigenic stimulation of HSAloB cellsin vitroleads to the generation of memory B-cell clones in the absence of any detectable antibody formation whereas stimulation of HSAint-hicells yield AFC responses but not memory B cells. Furthermore, the progeny of HSAlocells are unique in their ability to accumulate somatic mutations and originate germinal centers (GC). The pre-existence of two distinct precursor cell populations may help resolve the disparate biological characteristics of AFC precursors which appear to be terminally differentiated versus memory progenitors which retain stem cell characteristics in their capacity to self renew, undergo multiple divisions, and generate progeny that express enzymes characteristic of stem cells or pro-pre B cells and acquire tolerance susceptibility.

Activation of HLA-A2-specific memory B cells in severe combined immunodeficient mice

Peripheral blood leukocytes of all five HLA-A2-sensitized patients produced significant levels of human IgG (≥0.25 μg/ml) following polyclonal activation in vitro, but PBLs from only one patient (K.H.) who had been transfused recently (<4 weeks) produced detectable anti-HLA-A2 IgG. PBLs from this in vitro responder and from one in vitro nonresponder (L.G.) were transferred intraperitoneally into SCID mice. A low level (range, 5–40 ng/ml) of human anti-HLA-A2 IgG was detected in the serum of the mice without additional stimulation. This anti-HLA-A2 IgG response was boosted (range, 40–200 ng/ml) when mice received a human skin xenograft or an early challenge with x-irradiated human leukocytes intraperitoneally. Although the anti-HLA antibodies produced were specific for HLA-A2, the boosting of anti-HLA-A2 IgG production did not require the expression of the HLA-A2 protein, since either HLA-A2-negative skin xenografts or HLA-identical x-irradiated PBLs enhanced the production of anti-HLA-A2 IgG. Dose-response of transferred PBLs and κ:λ composition of individual mouse anti-HLA-A2 production suggested that low-frequency human memory B-cell clones were stimulated to proliferate and/or triggered to become high Ab secretors by skin graft or PBL boost.

Immunomorphological Characteristics of Human Peyer’s Patches

Human follicle-associated epithelium (FAE) was found not to express the secretory component (SC) or polymeric immunoglobulin (pIg) receptor, and is therefore unable to transport pIgA to the gut lumen. However, human FAE (except the M cells) was positive for MHC class II (HLA-DR) determinants. It may therefore perform class II-restricted uptake and presentation to T cells of antigens that have been adequately processed, whereas the function of the M cells is perhaps limited to transport of particulate or undergraded luminal antigens to subjacent antigen-presenting cells (APC). A large number of APC of the inter-digitating cell phenotype (positive for HLA-DR and S-100 protein) and relatively few L1-positive reactive macrophages were found in PP between the follicles and beneath the FAE, compared with villous mucosa. Also, there were significantly more intra- and subepithelial T cells in PP than in distant villi, and the T cells were concentrated adjacent to the M cells. The proportion of the CD4+ subset (putative helper T cells) was much higher in FAE (40%) than in villous epithelium where the CD8+ subset (putative suppressor T cells) predominated strikingly (90%). This disparity might reflect differences in capacity for positive and negative immune regulation at the two sites. The relatively few B cells terminating with Ig production in PP apparently belonged to relatively mature memory clones as they showed a large proportion of IgG immunocytes and reduced J-chain expression. Conversely, both IgG and IgA immunocytes in distant mucosa showed a high percentage of J-chain positivity (80-100%); such expression was also considerable (45-60%) in mesenteric lymph nodes (MLN) in contrast to peripheral lymph nodes (PLN) and palatine tonsils. Moreover, there was a decreasing percentage of IgA2 immunocytes in the order of PP (52%), distant ileal mucosa (40%), MLN (32%), PLN (11%), tand tonsils (5%). These results support the notion that migration of relatively immature memory B-cell clones takes place from PP through MLN preferentially to distant intestinal mucosa.

Human Peyer's Patches: Lympho-Epithelial Relationships and Characteristics of Immunoglobulin-Producing Cells

Human Peyers patches (PP) were studied by immunohistochemistry to characterize functional properties of the follicle-associated epithelium (FAE) including the "membrane" (M) cells. The FAE had no transporting capacity for polymeric IgA (pIgA) because it did not express the secretory component (SC) which acts as a pIgA receptor. However, it expressed MHC class II (HLA-DR) determinants, except for the M cells (which were tentatively identified by absence of brush border alkaline phosphatase). It is possible, therefore, that the FAE generally performs class II-restricted transport and presentation to T cells of antigens which have been adequately processed in the gut lumen. The function of M cells may be limited to transport of particulate or undegraded antigens to subjacent macrophages for processing and subsequent presentation. There were significantly more intra- and subepithelial T cells in PP than in distant villi, and the T cells were concentrated adjacent to M cells. The proportion of the CD4+ phenotype (putative helper T cells) was much higher in FAE (approximately 40%) than in villous epithelium where the CD8+ (putative suppressor) phenotype predominated strikingly (approximately 90%). This disparity might reflect differences in capacity for positive and negative immune regulation at the two sites. The B cells terminating with Ig production in PP and adjacent to solitary lymphoid follicles apparently belonged to relatively mature memory clones as they showed a large proportion of IgG immunocytes and reduced J-chain expression. Conversely, both IgG and IgA immunocytes in lamina propria (LP) showed a high percentage of J-chain positivity (80-100%); such positivity was also considerable (45-60%) in mesenteric lymph nodes (MLN) in contrast to peripheral lymph nodes (PLN) and palatine tonsils (PT). Moreover, there was a decreasing percentage of IgA2 immunocytes in the order of PP (52%), distant ileal LP (40%), MLN (32%), PLN (11%), and PT (5%). Taken together, our results suggested that dissemination of relatively immature memory B-cell clones with high J-chain expression takes place from PP through MLN and that preferential settlement of such clones occurs in LP.

B-cell memory is short-lived in the absence of antigen.

Primary encounter with antigen stimulates specific B cells not only to differentiate into cells that produce antibody at a high rate (plasma cells), but also to give rise to populations of memory cells. These cells have many characteristics that differ from virgin B cells, including their lifespan. When re-exposed to antigen, memory cells generate secondary IgG responses that are enhanced in rate, titre and affinity. At present they are considered as small resting lymphocytes which survive for long periods in a quiescent state between each antigen encounter. However, the fact that an individual may continue to make an antibody response for many months following a single injection of antigen is often overlooked. This continued antibody production is probably due to repeated stimulation of antigen-specific B cells and raises the question of whether memory B-cell clones require antigen for their maintenance. Here we show that they do, and that following transfer, in the absence of antigen, memory B-cell populations are lost from the adoptive host after 10-12 weeks.