Immunodominance of the VH1-46 antibody gene segment in the primary repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones

Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and VH gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19+IgD+CD27-, CD19+IgD+CD27+, or CD19+IgD-CD27+) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19+IgD+CD27+ unclass-switched memory B cells and a low frequency of somatic mutations in CD19+IgD-CD27+ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the VH1-46 gene segment was a prominent feature in the VH gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the VH gene repertoire of the two memory B cell groups derived from broader usage of VH gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease

Invasive pneumococcal infection is an important cause of morbidity and mortality in HIV-1-infected individuals. B cells play an important role in maintaining serologic memory after infection. IgM memory B cells are significantly reduced in HIV-1-infected patients and their frequency is similar to that observed in other patient groups (splenectomized individuals and patients with primary Ab deficiency) who are also known to have an increased risk of invasive pneumococcal infection. Antiretroviral therapy does not restore marginal zone B cell percentages. Immunization with the 23-valent polysaccharide pneumococcal vaccine shows that HIV-1-infected patients have impaired total IgM and IgG pneumococcal vaccines compared with healthy controls. Loss of switched memory B cells was associated with impaired tetanus toxoid IgG vaccine responses. Results of this study demonstrate that defects in B cell memory subsets are associated with impaired humoral immune responses in HIV-1 patients who are receiving antiretroviral therapy and may be a contributory factor to the increased risk of invasive pneumococcal infection observed in HIV-1 infection.     

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG⁺ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS⁺ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.     

VH gene analysis of spontaneously activated B cells in adult MRL-lpr/lpr mice. J558 bias is not limited to classic lupus autoantibodies

To determine the genetic origins of lupus auto-antibodies, we analyzed the relationship between VH gene usage and auto-Ag-binding properties of 352 B cell hybridomas derived from MRL-lpr/lpr mice. The hybridomas were derived from neonatal, 1-month-old, 3-month-old, and 6-month-old mice. The experimental strategy provided that the hybridomas were monoclonal at initial evaluation, so the Ag binding and V gene frequencies of the entire population could be determined. Initially, 1032 Ig-producing hybridomas were evaluated for binding to six Ag; VH gene family use was determined in 119 anti-DNA and anti-rabbit thymus extract (RTE) antibodies (autoantibodies) and in 233 age-matched Ig that did not bind to any of the six Ag (nonbinders). Neonatal B cells, including cross-reactive IgM autoantibodies and nonbinder IgM, used relatively 3' VH genes. The majority of B cells in adult mice used VH genes of the J558 family. Although J558 use was significantly higher among the autoantibodies (anti-DNA and anti-RTE) than among the nonbinder Ig, this difference was due to a higher frequency of J558 use by 1-month-old mice. At 3 months, J558 use by the nonbinder Ig increased to the same frequency of J558 use as in the autoantibody population. J558 use in both groups of antibodies exceeded a previously reported estimation of J558 expression in the functional B cell repertoire of young adult MRL-lpr/lpr mice. Several subgroups of antibodies that share properties with pathogenic Ig, including IgG, cross-reactive Ig, and anti-dsDNA autoantibodies, demonstrated a marked preferential expression of the J558 family. These results suggest that there is an age-related bias in the activation of B cells using J558 VH genes in MRL-lpr/lpr mice that is under the influence of a selective force distinct from, or in addition to, an ssDNA or RTE auto-Ag-driven response.     

The human antibody response to porcine xenoantigens is encoded by IGHV3-11 and IGHV3-74 IgVH germline progenitors.

Preformed and induced Ab responses present a major immunological barrier to the use of pig organs for human xenotransplantation. We generated IgM and IgG gene libraries established from lymphocytes of patients treated with a bioartificial liver (BAL) containing pig hepatocytes and used these libraries to identify IgVH genes that encode human Ab responses to pig xenoantigens. Genes encoded by the VH3 family are increased in expression in patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated, and the relative frequency of expression of genes used to encode the Ab response was determined at days 0, 10, and 21. Ig genes derived from the IGHV3-11 and IGHV3-74 germline progenitors increase in frequency post-BAL. The IGHV3-11 gene encodes 12% of VH3 cDNA clones expressed as IgM Abs at day 0 and 32.4-39.0% of cDNA clones encoding IgM Abs in two patients at day 10. IGHV3-11 and IGHV3-74 genes encoding IgM Abs in these patients are expressed without evidence of somatic mutation. By day 21, an isotype switch occurs and IGHV3-11 IgVH progenitors encode IgG Abs that demonstrate somatic mutation. We cloned these genes into a phagemid vector, expressed these clones as single-chain Abs, and demonstrated that the IGHV3-11 gene encodes Abs with the ability to bind to the gal alpha (1,3) gal epitope. Our results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline progenitors. IgM Abs are expressed in germline configuration and IgG Abs demonstrate somatic mutations by day 21.     

Both Memory and CD45RA+/CD62L+ Naive CD4+ T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.     

High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions

Celiac disease is an immune-mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2) are generated in response to the exogenous antigen gluten in individuals who express human leukocyte antigen HLA-DQ2 or HLA-DQ8 (ref. 3). We assessed in a comprehensive and nonbiased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire of ex vivo-isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are markedly expanded within the duodenal mucosa in individuals with active celiac disease. TG2-specific antibodies were of high affinity yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts, the TG2-specific ASCs had not recently proliferated and were not short-lived ex vivo. Altogether, these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favor massive generation of autoreactive B cells. TG2-specific antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naive IgD- and IgM-expressing B cells, thus possibly explaining why the antibody response to TG2 bears signs of a primary immune response despite the disease chronicity.     

Antibody repertoire development in fetal and neonatal piglets. XIII. Hybrid VH genes and the preimmune repertoire revisited

The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise approximately 80% of the preimmune repertoire. However, so-called "hybrid" VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied > 3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for > 95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.     

Regulation of IgM and IgD synthesis in B lymphocytes. II. Translational and post-translational events

Studies investigating the relative rates of biosynthesis of mu- and delta-polypeptide chains in normal resting B lymphocytes have shown that the translation rate of mu m is about sevenfold higher than that of delta m, thus reflecting the relative abundance of mRNA encoding these two chains. The turnover rate of cell surface IgM is faster, however, than that of cell surface IgD, resulting in higher expression of cell surface IgD relative to IgM under steady state conditions. LPS stimulation of B lymphocytes induces the complete cessation of synthesis of the delta-chain, thus accounting for the gradual disappearance of IgD from the cell surface of activated cells.     

IL-27 induces the production of IgG1 by human B cells

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human na?ve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to na?ve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by na?ve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.     

The IgM antibody level against ganglioside GM2 correlates to the disease status of HIV-1-infected patients

HIV-1 infection induces the expression of high level of GM2 ganglioside on infected cells and IgM antibody (Ab) against GM2 can cause complement (C)-mediated cytolysis of HIV-1-infected cells. Since GM2 is immunogenic in human, we proposed that an anti-GM2 IgM Ab may be produced by some HIV-1-infected patients and the titer of this Ab might provide some insight into the progress of the disease. On this premise, the amount of IgM Ab against GM2 was determined in 124 HIV-1-infected patients and 111 seronegative donors. As expected, the anti-GM2 IgM Ab titers of the patients was significantly higher than that of the seronegative donors while the total IgM levels remained unchanged. In addition, we determined the CD4+ cell count and the HIV-RNA load in the HIV-1-infected patients. The results showed a positive correlation between the anti-GM2 IgM Ab titer and CD4+ cell count but a negative correlation between the anti-GM2 IgM Ab titer and HIV-RNA load. These suggest that anti-GM2 IgM Ab induced and/or enhanced by HIV-1 infection causes C-mediated cytolysis of HIV-1-infected cells in vivo to a certain extent, and may help lower the plateau level of the HIV-RNA load. Therefore, the amount of IgM Ab against GM2 may be related to the prognosis of HIV-1 infected patients.