Three-dimensional genome rewiring during the development of antibody-secreting cells.

The development of B lymphocytes into antibody-secreting plasma cells is central to the adaptive immune system in that it confers protective and specific antibody response against invading pathogen. This developmental process involves extensive morphological and functional alterations that begin early after antigenic stimulation. These include chromatin restructuring that is critical in regulating gene expression, DNA rearrangement and other cellular processes. Here we outline the recent understanding of the three-dimensional architecture of the genome, specifically focused on its contribution to the process of B cell activation and terminal differentiation into antibody-secreting cells.

Targeting plasma cells: are we any closer to a panacea for diseases of antibody-secreting cells?

Antibody-secreting cells (ASCs) are critical for a functional and effective adaptive immune system. In a number of illnesses, however, these same cells contribute to the underlying disease state leading to significant morbidity and mortality. While therapeutic targeting of antibody-secreting cells has progressed significantly over the last two decades, many of these conditions remain major health problems. In this review, we will discuss current and potential therapeutic targeting of ASCs in the context of the known biology of these cells.

Stable long-term cultures of self-renewing B cells and their applications.

Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen-presenting cells for the discovery of tumor neoantigens.

Loss of Fancc Impairs Antibody-Secreting Cell Differentiation in Mice through Deregulating the Wnt Signaling Pathway.

Fanconi anemia (FA) is characterized by a progressive bone marrow failure and an increased incidence of cancer. FA patients have high susceptibility to immune-related complications such as infection and posttransplant graft-versus-host disease. In this study, we investigated the effect of FA deficiency in B cell function using the Fancc mouse model. Fancc(-/-) B cells show a specific defect in IgG2a switch and impaired Ab-secreting cell (ASC) differentiation. Global transcriptome analysis of naive B cells by mRNA sequencing demonstrates that FA deficiency deregulates a network of genes involved in immune function. Significantly, many genes implicated in Wnt signaling were aberrantly expressed in Fancc(-/-) B cells. Consistently, Fancc(-/-) B cells accumulate high levels of ß-catenin under both resting and stimulated conditions, suggesting hyperactive Wnt signaling. Using an in vivo Wnt GFP reporter assay, we verified the upregulation of Wnt signaling as a potential mechanism responsible for the impaired Fancc(-/-) B cell differentiation. Furthermore, we showed that Wnt signaling inhibits ASC differentiation possibly through repression of Blimp1 and that Fancc(-/-) B cells are hypersensitive to Wnt activation during ASC differentiation. Our findings identify Wnt signaling as a physiological regulator of ASC differentiation and establish a role for the Wnt pathway in normal B cell function and FA immune deficiency.

De Novo MS/MS Sequencing of Native Human Antibodies.

One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology, followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and represent only a small percentage of the total B-cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required without the need for sequencing of genetic material. Peripheral pAbs from a cytomegalovirus-exposed individual were purified by glycoprotein B antigen affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1 to 2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.

Natural IgM is produced by CD5- plasma cells that occupy a distinct survival niche in bone marrow.

Natural IgM is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. Natural IgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. Nevertheless, the origins of natural IgM have not been precisely defined. Previous studies focused on the role of CD5(+) B1 cells in the production of natural IgM, but we show in this article that a discrete population of CD5(-) IgM plasmablasts and plasma cells in the bone marrow (BM) produces the majority of serum IgM in resting mice. These Ab-secreting cells (ASC) originate from peritoneal cavity-resident cells, because transfer of peritoneal cells completely restores serum IgM and the specific compartment of BM ASC in Rag1-deficient mice. We show that BM natural IgM ASC arise from a fetal-lineage progenitor that is neither B1a nor B1b, and that this IgM ASC compartment contains a substantial fraction of long-lived plasma cells that do not occupy the IgG plasma cell survival niche in the BM; instead, they are supported by IL-5. In summary, we identified the primary source of natural IgM and showed that these ASC are maintained long-term in a unique survival niche within the BM.

Regulatory B10 cells differentiate into antibody-secreting cells after transient IL-10 production in vivo.

Regulatory B cells that are functionally defined by their capacity to express IL-10 (B10 cells) downregulate inflammation and autoimmunity. In studies using well-defined IL-10 reporter mice, this rare B10 cell subset was also found to maintain a capacity for plasma cell differentiation. During a transient period of il10 transcription, the blimp1 and irf4 transcription factors were induced in B10 cells, whereas pax5 and bcl6 were downregulated as a significant fraction of B10 cells completed the genetic and phenotypic program leading to Ab-secreting cell differentiation in vitro and in vivo. B10 cell-derived IgM reacted with both self- and foreign Ags, whereas B10 cells generated Ag-specific IgG in response to immunizations. Moreover, B10 cells represented a significant source of serum IgM and IgG during adoptive-transfer experiments and produced Ag-specific, polyreactive and autoreactive Ab specificities that were consistent with their expression of a diverse AgR repertoire. Thereby, B10 cells limit inflammation and immune responses by the transient production of IL-10, and may facilitate clearance of their eliciting Ags through an inherent capacity to quickly generate polyreactive and/or Ag-specific Abs during humoral immune responses.

Potent induction of antibody-secreting B cells by human dermal-derived CD14+ dendritic cells triggered by dual TLR ligation.

Targeting CD14(+) dermal-derived dendritic cells (DDCs) is a rational approach for vaccination strategies aimed at improving humoral immune responses, because of their natural ability to stimulate naive B cells. In this study, we show that CD14(+) DDCs express mRNA for TLRs 1-9, but respond differentially to single or paired TLR ligands. Compared to single ligands, some combinations were particularly effective at activating CD14(+) DDCs, as shown by enhanced expression of B cell stimulatory cytokines (IL-6, IL-10, and TNF-α) and more pronounced phenotypic maturation. These combinations were resiquimod (R-848) plus polyinosinic-polycytidylic acid [Poly(I:C)], R-848 plus LPS, Pam3CSK4 plus Poly(I:C), and LPS plus Poly(I:C). We also found that selected TLR ligand pairs [R-848 plus either LPS or Poly(I:C)] were superior to individual agents at boosting the inherent capacity of CD14(+) DDCs to induce naive B cells to proliferate and differentiate into CD27(+) CD38(+) B cells that secrete high levels of IgG and IgA. When treated with the same TLR ligand combinations, CD14(+) DDCs also promoted the differentiation of Th1 (IFN-γ-secreting) CD4(+) T cells, but not of Th2 or Th17 CD4(+) T cells. These observations may help to identify adjuvant strategies aimed at inducing protective immune responses to various pathogens, including but not limited to HIV-1.

Specialized proresolving mediators enhance human B cell differentiation to antibody-secreting cells.

The resolution of inflammation is an active and dynamic process critical in maintaining homeostasis. Newly identified lipid mediators have been recognized as key players during the resolution phase. These specialized proresolving mediators (SPM) constitute separate families that include lipoxins, resolvins, protectins, and maresins, each derived from essential polyunsaturated fatty acids. New results demonstrate that SPM regulate aspects of the immune response, including reduction of neutrophil infiltration, decreased T cell cytokine production, and stimulation of macrophage phagocytic activity. The actions of SPM on B lymphocytes remain unknown. Our study shows that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA), resolvin D1, and protectin D1 are present in the spleen. Interestingly, 17-HDHA and resolvin D1, but not protectin D1, strongly increase activated human B cell IgM and IgG production. Furthermore, increased Ab production by 17-HDHA is due to augmented B cell differentiation toward a CD27(+)CD38(+) Ab-secreting cell phenotype. The 17-HDHA did not affect proliferation and was nontoxic to cells. Increase of plasma cell differentiation and Ab production supports the involvement of SPM during the late stages of inflammation and pathogen clearance. The present study provides new evidence for SPM activity in the humoral response. These new findings highlight the potential applications of SPM as endogenous and nontoxic adjuvants, and as anti-inflammatory therapeutic molecules.

Plasma cell S1P1 expression determines secondary lymphoid organ retention versus bone marrow tropism.

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

TLR9 drives the development of transitional B cells towards the marginal zone pathway and promotes autoimmunity.

Maturation of B cells depends on environmental stimuli. Peripheral immature B cells develop into follicular pathway when antigenic stimulation is combined with T cell signals. Here, we wished to identify stimuli contributing to the development into marginal zone B cells known to be involved in autoimmune response. We found that TLR9 stimulation of transitional B cells induces proliferation and specific maturation into CD24(-) CD38(+) CD21(high) CD23(low) IgM(high) IgD(low) and Notch2(high) B cells characteristics of marginal zone B cells. Terminal differentiation into antibody-secreting cell associated with isotype switch commitment is also triggered which leads to a striking production of autoantibodies. Interestingly, mature B cells do not differentiate into marginal zone pathway following TLR9 stimulation, nor do transitional B cells under antigenic and T cell combined signals. These results suggest that transitional B cells are specifically sensitive to TLR9 stimulation to produce autoreactive marginal zone B cells.

[Effect of radiation-induced bystander chemosignals of mice on the humoral immune response in spleen and lymph nodes of intact recipients].

The ability of post-radiation (4 Gy) bystander chemosignals (the volatile components of mouse urine) to distantly modulate the humoral immune response to the sheep red blood cells in the spleen and popliteal lymph nodes of intact recipients has been investigated. It was shown that the exposure of animals to chemosignals before antigen injection resulted in the decrease and increase of the immune response in the spleen and lymph nodes, respectively. When animals were exposed to chemosignals after the antigenic stimulus, an increased immune response was observed in both spleen and lymph nodes. The contribution of radiation-induced bystander signaling in the response of socially organized animals to the effect of ionizing irradiation is discussed.

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

Supplemental ß-carotene increases IgA-secreting cells in mammary gland and IgA transfer from milk to neonatal mice.

Mortality of neonates continues to be a major problem in humans and animals. IgA provides protection against microbial antigens at mucosal surfaces. Although ß-carotene supplementation has been expected to enhance retinoic acid-mediated immune response in neonates, the exact mechanism by which ß-carotene enhances IgA production is still unclear. We investigated the effect of supplemental ß-carotene for maternal mice during pregnancy and lactation on IgA antibody-secreting cells (ASC) in mammary gland and guts and on IgA transfer from milk to neonatal mice. Pregnant mice were fed untreated or 50 mg/kg ß-carotene-supplemented diets from 6·5 d postcoitus (dpc) to 14 d postpartum (dpp). Supplemental ß-carotene increased the numbers of IgA ASC in mammary gland (P < 0·05) and ileum (P < 0·001), and also mRNA expression of IgA C-region in ileum (P < 0·05) of maternal mice at 14 dpp, but few IgA ASC were detected in mammary gland at 17·5 dpc. IgA concentration in stomach contents, which represents milk IgA level, was significantly higher (P < 0·01) in neonatal mice born to ß-carotene-supplemented mothers at 7 and 14 dpp, and IgA concentration in serum, stomach contents and faeces increased (P < 0·001) drastically with age. These results suggest that ß-carotene supplementation for maternal mice during pregnancy and lactation is useful for enhancing IgA transfer from maternal milk to neonates owing to the increase in IgA ASC in mammary gland and ileum during lactation.

Sublingual immunization with nonreplicating antigens induces antibody-forming cells and cytotoxic T cells in the female genital tract mucosa and protects against genital papillomavirus infection.

We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses. In this study, the potential of s.l. immunization to induce remote Ab responses and CD8(+) cytotoxic responses in the female genital tract was examined in mice by using a nonreplicating Ag, OVA, and cholera toxin (CT) as an adjuvant. Sublingual administration of OVA and CT induced Ag-specific IgA and IgG Abs in blood and in cervicovaginal secretions. These responses were associated with large numbers of IgA Ab-secreting cells (ASCs) in the genital mucosa. Genital ASC responses were similar in magnitude and isotype distribution after s.l., intranasal, or vaginal immunization and were superior to those seen after intragastric immunization. Genital, but not blood or spleen, IgA ASC responses were inhibited by treatment with anti-CCL28 Abs, suggesting that the chemokine CCL28 plays a major role in the migration of IgA ASC progenitors to the reproductive tract mucosa. Furthermore, s.l. immunization with OVA induced OVA-specific effector CD8(+) cytolytic T cells in the genital mucosa, and these responses required coadministration of the CT adjuvant. Furthermore, s.l. administration of human papillomavirus virus-like particles with or without the CT adjuvant conferred protection against genital challenge with human papillomavirus pseudovirions. Taken together, these findings underscore the potential of s.l. immunization as an efficient vaccination strategy for inducing genital immune responses and should impact on the development of vaccines against sexually transmitted diseases.

Diagnosis of Streptococcus pneumoniae infection using circulating antibody secreting cells.

BACKGROUND: Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes. METHODS: We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients. RESULTS: We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative. CONCLUSION: Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

Suppression of reaginic antibody (IgE) formation in mice by treatment with anti-mu antiserum.

Neonatally initiated injection of anti-mu antiserum in mice has been shown to suppress the formation of reaginic antibodies in response to infection with the intestinal nematode, Nippostrongylus brasiliensis. This observation supports the hypothesis that IgE-producing cells arise from IgM-bearing precursors.

Immunologic memory cells of bone marrow origin. Increased burst size of specific immunocyte precursors.

Individual immunocompetent precursor cells of (C57BL/10 x C3H)F(1) mouse marrow generate, on transplantation, three to five times more antibody-forming cells localized in recipient spleens during secondary than during primary immune responses. The increased burst size is immunologically specific since antigens of horse and chicken erythrocytes and of Salmonella typhimurium do not cause this effect in marrow cells responsive to sheep red blood cells. Both sensitized and nonsensitized precursors require the helper function of thymus-derived cells and antigen for the final steps of differentiation and maturation. The burst size of primed precursor cells is the same after cooperative interactions with virgin or educated helper cells of thymic origin. The greater potential of these marrow precursors may be attributable to self-replication and migration before differentiation into antibody-forming descendants. In fact, the progeny cells of primed precursor units are distributed among a multiplicity of foci, whereas those of nonimmune precursors are clustered into one focus. The described properties of specifically primed marrow precursors are those underlying immunologic memory. It remains to be established whether memory cells are induced or selected by antigens and whether the thymus plays a role in this process.

Studies on antibody-producing cells. IV. Ultrastructue of plaque-forming cells of rabbit lymph.

Efferent lymph of the popliteal lymph nodes of rabbits was collected 4 days after a single footpad injection of SRBC. Thin-layer agar plating was done to isolate plaque-forming cells of the lymph for electron microscope examination, and the numbers of plaque-forming cells (PFC) in cells from the lymph and lymph nodes were determined. Of 71 PFC of lymph isolated and examined, 93% were lymphocytes, most of them with signs of substantial levels of physiologic activity. The cytoplasm showed an abundance of free ribosomes and many finger-like projections. The endoplasmic reticulum (ER) was barely detectable in most of the active lymphocytic PFC, and in some, a few short narrow channels of ER could be seen. Approximately one-fifth of the lymphocytic PFC presented an appearance of senescence, with signs of degeneration: rounded cells, with amorphous nuclear chromatin, and very few microvilli. The remaining 7% of the PFC of the lymph showed an unusual combination of features: small round cells with a narrow ring of cytoplasm which, however, contained well-organized channels of ER. Such cells had been found only among PFC of peripheral blood of the rabbit. The number of PFC per million cells was higher in the lymph than in the suspensions of lymph node cells. In both the contralateral lymph node and its efferent lymph, the number of PFC was less than 1% that of the injected side.