Human monoclonal antibodies by immortalization of memory B cells

The administration of hyper immune sera to prevent or treat life-threatening infections is a remarkable milestone in medicine and biotechnology that has been achieved more than a century ago. Yet, the therapeutic use of monoclonal antibodies in this field has developed slowly over the last decades. Here we compare and contrast current methods to generate human monoclonal antibodies and highlight the advantages of exploiting the human antibody repertoire using a novel method that allows efficient immortalization and cloning of human memory B cells. This method, which has been successfully applied to isolate broadly neutralizing antibodies against SARS and H5N1 influenza viruses, is expected to accelerate the development of therapeutics in the field of infectious diseases not only by providing neutralizing antibodies for passive serotherapy, but also by generating relevant information for vaccine design.     

Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: role of memory B cells and CD5+ B cells.

NK cells are a subpopulation of lymphocytes characterized primarily by their cytolytic activity. They are recognized as an important component of the immune response against virus infection and tumors. In addition to their cytolytic activity, NK cells also participate either directly or indirectly in the regulation of the ongoing Ab response. More recently, it has been suggested that NK cells have an important role in the outcome of autoimmune diseases. Here, we demonstrate that human NK cells can induce autologous resting B cells to synthesize Ig, including switching to IgG and IgA, reminiscent of a secondary Ab response. B cell activation by the NK cell is contact-dependent and rapid, suggesting an autocrine B cell-regulated process. This NK cell function is T cell-independent, requires an active cytoplasmic membrane, and is blocked by anti-CD40 ligand (anti-CD154) or CD40-mIg fusion protein, indicating a critical role for CD40-CD40 ligand interaction. Depletion studies also demonstrate that CD5+ B cells (autoreactive B-1 cells) and a heterogeneous population of CD27+ memory B cells play a critical role in the Ig response induced by NK cells. The existence of this novel mechanism of B cell activation has important implications in innate immunity, B cell-mediated autoimmunity, and B cell neoplasia.     

B memory cells in the thymus: part of the pool of potentially circulating memory cells.

Immunization of mice with sheep red blood cells (SRBC) or Escherichia coli lipopolysaccharide (LPS) induces the appearance of B memory cells in the thymus. In this paper the origin of these B memory cells was investigated. Therefore, mice primed with either SRBC or LPS 6 months previously and nonprimed mice were joined for parabiosis. Four weeks later the parabiotic mice were separated from each other. Another 3 weeks later thymus cells from the primed and nonprimed mice were transferred separately into lethally irradiated mice in order to determine the adoptive PFC response. It was found that the 4-week period of parabiosis could account for the appearance of a distinct population of B memory cells in the thymus of the nonprimed mice. This result suggest that the B memory cells which appear in the thymus belong to the pool of potentially circulating memory cells.     

Silent development of memory progenitor B cells

T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.     

Cancer cells and parasites: two of a kind

Cancer cells and parasites use similar molecular mechanisms for survival in their hosts. Indeed, cancer cells might be thought of as normal cells that have lost their growth control mechanisms and have acquired parasitic features.     

Identification of memory B cells using a novel transgenic mouse model

Memory B cells help to protect the host from invading pathogens by maintaining persistent levels of Ag-specific serum Ab and generating rapid Ab responses upon re-exposure to Ag. Unambiguous identification of memory B cells has been a major obstacle to furthering our knowledge concerning both the development of B cell memory and secondary Ab responses due to an absence of specific cell surface markers. Germinal centers (GCs) are thought to be the major site of Ig hypermutation and Ag-driven selection of memory B cells. To develop a model that would identify GC-derived memory B cells, we generated transgenic mice that expressed cre recombinase in a GC-specific fashion. Interbreeding these mice with the cre-reporter strain, ROSA26R, produced progeny in which beta-galactosidase (beta-gal) was permanently expressed in B cells of the GC-memory pathway. Analysis following immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma globulin showed that long-lived beta-gal+ B cells exclusively contained somatically mutated lambda1 V regions and were capable of producing Ag-specific Ab-forming cell (AFC) responses that were >100-fold higher than those afforded by beta-gal- B cells following adoptive transfer to naive hosts. Secondary challenge of immune mice showed that only approximately 20% of secondary AFCs expressed beta-gal. Interestingly, we found that somatic hypermutation of rearranged lambda1 V regions within secondary AFCs showed a strong correlation with beta-gal expression, suggesting that nonmutated B cells contribute significantly to secondary Ab responses. This model should provide useful insights into memory B cell development, maintenance, and differentiation following immunization or pathogenic infection.     

Preparation and analysis of antigen-specific memory B cells

A procedure has been developed for the enrichment of TNP-binding memory B cells (TNP-MABC) from spleens of immunized mice. More than 75% of the cells expressed surface IgM (sIgM) and IgD (sIgD) and about 9% expressed surface IgG (sIgG). The TNP-MABC consisted of small resting lymphocytes with high affinity antigen-binding receptors. These cells expressed increased densities of Ia antigens and decreased densities of sIgD. Adoptive transfer of the cells into irradiated, carrier-primed syngeneic recipients resulted in their differentiation into IgG anti-TNP antibody-secreting cells. TNP-MABC secreted high affinity IgG anti-TNP antibodies when cultured in vitro with carrier-primed T cells and antigen. Limiting dilution analysis revealed that TNP-MABC contained a relatively low frequency of precursors for IgG-secreting cells that had an exceptionally large clone size. These results show that a highly enriched population of antigen-specific memory B cells can now be prepared and used to analyze their activation requirements.     

Tolerance susceptibility of newly generating memory B cells

Newly generating memory B cells rapidly accumulate somatic mutations that can alter their Ag-combining sites and potentially engender recognition of self determinants. To investigate the possibility that, during their emergence secondary B cells pass through a window of tolerance susceptibility, we have examined the in vitro generation of memory B cells in the presence or absence of tolerogen. The findings indicate that, before antigenic stimulation, precursors to memory B cells are resistant to tolerance induction. However, 2 to 7 days after T cell-dependent antigenic stimulation, newly emerging hapten-specific secondary B cells can be inactivated by the presence of hapten on a carrier not recognized by available Th cells. This inactivation can be blocked by the presence of free hapten and can be competed by the presence of immunogen. Inactivation of newly generating secondary B cells appears less specific than the tolerance induction of immature neonatal or bone marrow B cells because inactivation can be accomplished by cross-reactive determinants. Interestingly, the presence of tolerogen after primary stimulation did not preclude the generation of cells responsive to a third in vitro stimulation. Therefore, whereas newly emerging memory B cells are highly susceptible to inactivation, the progression of the clones of progenitors to memory B cells appears resistant to tolerance induction.     

Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting state.

Traditionally, emphasis has been placed on the roles of Th cells in generating and amplifying both cellular and humoral memory responses. Little is known about the potential contributions of B cell subsets to immunological memory. Resting memory B cells have generally been regarded as poor APC, attributed in part to the relative paucity of costimulatory molecules identified on their surface. We describe a novel subpopulation of human memory B cells that express CD80 in their resting state, are poised to secrete particularly large amounts of class switched Igs, and can efficiently present Ag to and activate T cells. This functionally distinct B cell subset may represent an important mechanism by which quiescent human B cells can initiate and propagate rapid and vigorous immune memory responses. Finally, these studies extend recent observations in the murine system and highlight the phenotypic and functional diversity that exists within the human B cell memory compartment.     

Granzyme B production distinguishes recently activated CD8(+) memory cells from resting memory cells

For immune diagnostic purposes it would be critical to be able to distinguish between ongoing immune processes, such as active infections, and long-term immune memory, for example imprinted by infections that have been cleared a long time ago or by vaccinations. We tested the hypothesis that the secretion of granzyme B, as detected in ex vivo ELISPOT assays, permits this distinction. We studied EBV-, flu- and CMV-specific CD8(+) cells in healthy individuals, Vaccinia virus-reactive CD8(+) cells in the course of vaccination, and HIV-specific CD8(+) cells in HIV-infected individuals. Antigen-specific ex vivo GzB production was detected only transiently after Vaccinia immunization, and in HIV-infected individuals. Our data suggest that ex vivo ELISPOT measurements of granzyme B permit the identification of actively ongoing CD8(+) cell responses-a notion that is pertinent to the immune diagnostic of infections, transplantation, allergies, autoimmune diseases, tumors and vaccine development.     

Systematic comparison of gene expression between murine memory and naive B cells demonstrates that memory B cells have unique signaling capabilities

Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naive precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of Ag-specific memory and naive cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. We identified genes with differential expression and confirmed the differential expression of many of these by quantitative RT-PCR and of some of these at the protein level. Our initial analysis revealed differential expression patterns of genes that regulate signaling. Memory B cells have increased expression of genes important in regulating adenosine signaling and in modulating cAMP responses. Furthermore, memory B cells up-regulate receptors that are essential for embryonic stem cell self-renewal. We further demonstrate that one of these, leukemia inhibitory factor receptor, can initiate functional signaling in memory B cells whereas it does not in naive B cells. Thus, memory and naive B cells are intrinsically wired to signal differently from one another and express a functional signaling pathway that is known to maintain stem cells in other lineages.     

Human C3 deficiency associated with impairments in dendritic cell differentiation, memory B cells, and regulatory T cells

Primary C3 deficiency, a rare autosomal inherited disease (OMIM 120700), was identified in a 2-year-old male suffering from recurrent pyogenic infections from early infancy with undetectable total complement hemolytic activity (CH50) and C3 values. The nonconsanguineous parents and the two patients' two siblings had 50% normal serum C3 concentration. The molecular abnormality associated a paternal allele coding C3 with the missense mutation p.Ser(550)Pro and an apparently null maternal allele, with production of a defective protein that could no longer be secreted. Vaccination of the child did not induce a long-term Ab response. Accordingly, switched memory IgD(-)CD27(+) B cells were barely detected, amounting to only 2.3% of peripheral blood CD19(+) cells. Cells were significantly defective in stimulating alloreactive responses. The in vitro development of immature dendritic cells and their maturation capacity were greatly impaired, with decreased CD1a expression and IL-12p70 secretion ability. These cells were unable to induce autologous B cell proliferation and Ig secretion in the presence of CD40L and C3. Finally, the regulatory T cell development ability of CD4(+) T cells after CD3 and CD46 activation in the presence of IL-2 was significantly impaired. Thus, the association of important functional defects of dendritic cells, acquisition of B cell memory, and regulatory T cells with human C3 deficiency strongly supports a major role for C3 in bridging innate and adaptive immunity in humans.     

Contributions of memory B cells to secondary immune response

The secondary immune response is one of the most important features of immune systems. During the secondary immune response, the immune system can eliminate the antigen, which has been encountered by the individual during the primary invasion, more rapidly and efficiently. Both T and B memory cells contribute to the secondary response. In this paper, we only concentrate on the functions of memory B cells. We explore a model describing the memory contributed by the specific long-lived clone which is maintained by continued stimulation with a small amount of antigens sequestered on the surfaces of the follicular dendritic cells (FDC). The behavior of the secondary response provided by the model can be compared with experimental observations. The model shows that memory B cells indeed play an important role in the secondary response. It is found that a single memory cell in a long-lived clone may not be long-lived. In the present note, the influences of relevant parameters on the secondary response are also explored.     

Role of B cells in maintaining helper T-cell memory

The cellular interactions involved in maintaining CD4+ T-cell memory have hitherto not been identified. In this report, we have investigated the role played by B cells in this process. We show that that long-lasting helper T-cell memory depends on the presence of B cells, but that direct antigen presentation by B cells is not required. These findings provide new insights into the mechanisms which underlie helper T-cell memory. They also suggest that the efficacy of future vaccines will depend critically on the inclusion of B- as well as T-cell epitopes.     

In vitro activation of murine antigen-specific memory B cells by a T-dependent antigen

A procedure that generates an enriched population (60 to 85%) of memory B cells specific for TNP (TNP-MABC) was employed. The activation requirements of TNP-MABC for the T-dependent antigen TNP-KLH and carrier-primed helper T (Th) cells were compared to those for TNP-binding cells from nonimmune mice (TNP-ABC). Proliferation and differentiation of TNP-MABC in response to cognate recognition of antigen requires less antigen and fewer carrier-primed Th cells than the activation of TNP-ABC. Furthermore, responses of the TNP-MABC were of a greater magnitude. Non-cognate activation induces a low level of proliferation of both TNP-ABC and TNP-MABC, but induces differentiation of TNP-MABC only. Percoll density fractionation of spleen cells prior to enrichment for TNP-MABC suggests that the small, dense cell population responds to cognate, but not to non-cognate activation. FACS separation of TNP-MABC by surface Ig isotype reveals that approximately 80% of the secondary IgG response is derived from cells expressing sIgG. Such cells constitute less than 10% of the total number of TNP-MABC. Limiting dilution studies with sorted TNP-MABC indicate that sIgG+ TNP-MABC are enriched for precursors that give rise to a large clone size. The in vitro results indicate the existence of three putative pathways for antigen-specific memory B cell activation by a T-dependent antigen: 1) sIgG+ cells differentiating into IgG-secreting cells; 2) sIgM+ cells differentiating into IgG-secreting cells; and 3) sIgM+ cells differentiating into IgM-secreting cells.     

Role of MHC class II on memory B cells in post-germinal center B cell homeostasis and memory response

We investigated the role of B cell Ag presentation in homeostasis of the memory B cell compartment in a mouse model where a conditional allele for the beta-chain of MHC class II (MHC-II) is deleted in the vast majority of all B cells by cd19 promoter-mediated expression of Cre recombinase (IA-B mice). Upon T cell-dependent immunization, a small number of MHC-II(+) B cells in IA-B mice dramatically expanded and restored normal albeit delayed levels of germinal center (GC) B cells with an affinity-enhancing somatic mutation to Ag. IA-B mice also established normal levels of MHC-II(+) memory B cells, which, however, subsequently lost MHC-II expression by ongoing deletion of the conditional iab allele without significant loss in their number. Furthermore, in vivo Ag restimulation of MHC-II(-) memory B cells of IA-B mice failed to cause differentiation into plasma cells (PCs), even in the presence of Ag-specific CD4(+) T cells. In addition, both numbers and Ag-specific affinity of long-lived PCs during the late post-GC phase, as well as post-GC serum affinity maturation, were significantly reduced in IA-B mice. These results support a notion that MHC-II-dependent T cell help during post-GC phase is not absolutely required for the maintenance of memory B cell frequency but is important for their differentiation into PCs and for the establishment of the long-lived PC compartment.