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.

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.

Rainbow trout mount a robust specific immune response upon anal administration of thymus-independent antigens.

Despite the strong demand for orally-delivered fish vaccines and the deficient response of those currently available in the market, little is known about how teleost B cells differentiate to antibody secreting cells (ASCs) in response to antigens delivered to the intestinal mucosa. To fill this gap, in the current study, we have studied the dynamics of B cell differentiation in spleen and kidney of rainbow trout (Oncorhynchus mykiss) anally immunized with antigens catalogued in mammals as thymus dependent (TD) or thymus-independent (TI). Our results show that, in the absence of additional adjuvants, rainbow trout preferentially responded to a model TI antigen such as TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The anal administration of TNP-LPS elicited TNP-specific serum antibodies, and a significant increase in the number of total and TNP-specific ASCs in both spleen and kidney, being the kidney the site where most ASCs are found at later time points. In the spleen, a proliferative response of both IgM+ B and T cells was also clearly visible, while the proliferative response was weaker in the kidney. Finally, TNP-LPS also provoked a transcriptional regulation of some immune genes in the spleen and the intestine, including a decreased transcription of foxp3a and foxp3b in intestine that suggests a breach in tolerogenic responses in response to TI stimulation. These results contribute to a better understanding of how intestinal immunity is regulated in teleost and will aid in the future design of effective oral strategies for aquaculture.

CD19-positive antibody-secreting cells provide immune memory.

Long-lived antibody-secreting cells (ASCs) are critical for the maintenance of humoral immunity through the continued production of antibodies specific for previously encountered pathogen or vaccine antigens. Recent reports describing humoral immune memory have suggested the importance of long-lived CD19- bone marrow (BM) ASCs, which secrete antibodies recognizing previously encountered vaccine antigens. However, these reports do not agree upon the unique contribution of the CD19+ BM ASC subset toward humoral immunity. Here, we found both CD19+ and negative ASCs from human BM were similar in functional capacity to react to a number of vaccine antigens via ELISpot assays. The CD19+ cells were the predominant ASC population found in lymphoid tissues, and unlike the CD19- ASCs, which were found only in spleen and BM, the CD19+ ASCs were found in tonsil and blood. CD19+ ASCs from the BM, spleen, and tonsil were capable of recognizing polio vaccine antigens, indicating the CD19+ ASC cells play a novel role in long-lasting immune defense. Comparative gene expression analysis indicated CD19+ and negative BM ASCs differed significantly by only 14 distinct messenger RNAs and exhibited similar gene expression for cell cycle, autophagy, and apoptosis control necessary for long life. In addition, we show identical CDR-H3 sequences found on both BM ASC subsets, indicating a shared developmental path. Together, these results provide novel insight for the distribution, function, genetic regulation, and development of long-lived ASCs and may not only impact improved cell therapies but also enhance strategies for vaccine development.

Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion.

Human antibody-secreting cells (ASC) in peripheral blood are found after vaccination or infection but rapidly apoptose unless they migrate to the bone marrow (BM). Yet, elements of the BM microenvironment required to sustain long-lived plasma cells (LLPC) remain elusive. Here, we identify BM factors that maintain human ASC > 50 days in vitro. The critical components of the cell-free in vitro BM mimic consist of products from primary BM mesenchymal stromal cells (MSC), a proliferation-inducing ligand (APRIL), and hypoxic conditions. Comparative analysis of protein-protein interactions between BM-MSC proteomics with differential RNA transcriptomics of blood ASC and BM LLPC identify two major survival factors, fibronectin and YWHAZ. The MSC secretome proteins and hypoxic conditions play a role in LLPC survival utilizing mechanisms that downregulate mTORC1 signaling and upregulate hypoxia signatures. In summary, we identify elements of the BM survival niche critical for maturation of blood ASC to BM LLPC.

Site-1 protease function is essential for the generation of antibody secreting cells and reprogramming for secretory activity.

The unfolded protein response (UPR) and activation of XBP1 is necessary for high secretory efficiency and functional differentiation of antibody secreting cells (ASCs). The UPR additionally includes a branch in which membrane-bound transcription factors, exemplified by ATF6, undergo intramembrane-proteolysis by the sequential action of site-1 (MBTPS1/S1P) and site-2 proteases (MBTPS2/S2P) and release of the cytoplasmic domain as an active transcription factor. Such regulation is shared with a family of CREB3-related transcription factors and sterol regulatory element-binding proteins (SREBPs). Of these, we identify that the CREB3 family member CREB3L2 is strongly induced and activated during the transition from B-cell to plasma cell state. Inhibition of site-1 protease leads to a profound reduction in plasmablast number linked to induction of autophagy. Plasmablasts generated in the presence of site-1 protease inhibitor segregated into CD38high and CD38low populations, the latter characterized by a marked reduction in the capacity to secrete IgG. Site-1 protease inhibition is accompanied by a distinctive change in gene expression associated with amino acid, steroid and fatty acid synthesis pathways. These results demonstrate that transcriptional control of metabolic programs necessary for secretory activity can be targeted via site-1 protease inhibition during ASC differentiation.

High-affinity IgM+ memory B cells are defective in differentiation into IgM antibody-secreting cells by re-stimulation with a T cell-dependent antigen.

IgM antibodies (Abs) are thought to play a major role in humoral immunity but only at the early stage of the primary immune response. However, two subsets of IgM+ memory B cells (MBCs), one with high affinity gained by means of multiple somatic hypermutation (SHM) and the other with low affinity and no SHMs, are generated through the germinal center (GC)-dependent and GC-independent (non-GC) pathway, respectively, after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin. Surprisingly, an analysis of antibody-secreting cells reveals that a large amount of anti-NP IgM Ab with few SHMs is secreted during the recall response, indicating that only non-GC MBCs have terminal differentiation potential. Since secondary IgM Abs are capable of binding to dinitrophenyl ligands, they likely provide broad cross-reactivity in defense against microbial infection.

Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells.

Immunological prevention of infectious disease, especially viral, is based on antigen-specific long-lived memory B cells. To test for cellular proliferation and differentiation factors in swine, an outbred model for humans, CD21+ B cells were activated in vitro with CD40L and stimulated with purported stimulatory cytokines to characterize functional responses. IL-21 induced a 3-fold expansion in total cell numbers with roughly 15% of all B cells differentiating to IgM or IgG antibody secreting cells (ASCs.) However, even with robust proliferation, cellular viability rapidly deteriorated. Therefore, a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF) were evaluated as survival and maintenance factors. BAFF was effective at enhancing the viability of mature B cells as well as ASCs, while APRIL was only effective for ASCs. Both cytokines increased approximately two-fold the amount of IgM and IgG which was secreted by IL-21 differentiated ASCs. Mature B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve age-matched pigs were activated and treated with IL-21 and then tested for memory cell differentiation using a PRRSV non-structural protein 7 ELISPOT and ELISA. PRRSV immune pigs were positive on both ELISPOT and ELISA while naïve animals were negative on both assays. These results highlight the IL-21-driven expansion and differentiation of memory B cells in vitro without stimulation of the surface immunoglobulin receptor complex, as well as the establishment of a defined memory B cell culture system for characterization of vaccine responses in outbred animals.

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.

The distribution of SIgA and IgG antibody-secreting cells in the palatine tonsils of Bactrian camels (Camelus bactrianus) of different ages.

Secretory immunoglobulin A (SIgA) antibody-secreting cells (ASCs) are the major effector cells of mucosal immunity, and immunoglobulin G (IgG) ASCs are also associated with mucosal immunity. This study aimed to explore the distribution of these 2 ASC populations in the palatine tonsils of Bactrian camels of different ages. Eighteen Bactrian camels were divided into the following three age groups: pubertal (3-5 years), middle-aged (6-16 years) and old (17-20 years). SIgA and IgG ASCs within different sites of the palatine tonsils were observed through histological and immunohistochemical techniques, and their densities were analyzed using statistical methods. The results from all age groups showed that both the SIgA and IgG ASCs were primarily distributed in the subepithelial compartments of the reticulated crypt epithelium and secondarily distributed in the subepithelial compartments of the stratified surface squamous epithelium, with a few ASCs located in the extrafollicular region. Their densities in these three areas were significantly decreased in turn (P<0.05). However, the densities of SIgA ASCs were significantly higher than IgG ASCs in the same regions (P<0.05), and the densities of both ASC populations decreased with age. The results confirmed that Bactrian camel palatine tonsils are the primary mucosal immune organ producing SIgA ASCs, and the subepithelial compartment of the reticulated crypt epithelium is the primary region for the colonization and functional activity of SIgA and IgG ASCs.

Age-related changes in the transcriptome of antibody-secreting cells.

We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Further analysis showed upon aging defects in energy production through glucose catabolism with reduced oxidative phosphorylation. In addition aged B cells had increased levels of reactive oxygen-species (ROS), which was linked to enhanced expression of the co-inhibitor programmed cell death (PD)-1.

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.

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.

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.

Immune reactivity in rats selected for the enhancement or elimination of aggressiveness towards humans.

This study analyzes immune reactivity in two lines of rats selected for the enhancement or elimination of aggressiveness toward humans. Compared to nonaggressive line, aggressive rats showed increased blood ratio of CD4(+) and CD8(+)T lymphocytes, monocyte chemoattractant protein (MCP)-1 level both before and after immunization with sheep red blood cells (SRBC), enhanced IgM-immune response, as well as decreased level of interleukin (IL)-1α before immunization. However, antigen administration produced IL-1α increase in aggressive rats and its decrease in nonaggressive rats compared to non-immunized rats of the same lines. In addition, line-dependent alterations of T lymphocyte distribution in response to immune activation have been found only in the spleen. It is suggested that genetic differences in aggressive behavior may contribute to differences in immune function.

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.

Modeling the dynamics and migratory pathways of virus-specific antibody-secreting cell populations in primary influenza infection.

The B cell response to influenza infection of the respiratory tract contributes to viral clearance and establishes profound resistance to reinfection by related viruses. Numerous studies have measured virus-specific antibody-secreting cell (ASC) frequencies in different anatomical compartments after influenza infection and provided a general picture of the kinetics of ASC formation and dispersion. However, the dynamics of ASC populations are difficult to determine experimentally and have received little attention. Here, we applied mathematical modeling to investigate the dynamics of ASC growth, death, and migration over the 2-week period following primary influenza infection in mice. Experimental data for model fitting came from high frequency measurements of virus-specific IgM, IgG, and IgA ASCs in the mediastinal lymph node (MLN), spleen, and lung. Model construction was based on a set of assumptions about ASC gain and loss from the sampled sites, and also on the directionality of ASC trafficking pathways. Most notably, modeling results suggest that differences in ASC fate and trafficking patterns reflect the site of formation and the expressed antibody class. Essentially all early IgA ASCs in the MLN migrated to spleen or lung, whereas cell death was likely the major reason for IgM and IgG ASC loss from the MLN. In contrast, the spleen contributed most of the IgM and IgG ASCs that migrated to the lung, but essentially none of the IgA ASCs. This finding points to a critical role for regional lymph nodes such as the MLN in the rapid generation of IgA ASCs that seed the lung. Results for the MLN also suggest that ASC death is a significant early feature of the B cell response. Overall, our analysis is consistent with accepted concepts in many regards, but it also indicates novel features of the B cell response to influenza that warrant further investigation.

Immune responses to O-specific polysaccharide and lipopolysaccharide of Vibrio cholerae O1 Ogawa in adult Bangladeshi recipients of an oral killed cholera vaccine and comparison to responses in patients with cholera.

Protective immunity to cholera is serogroup specific, and serogrouping is defined by the O-specific polysaccharide (OSP) of lipopolysaccharide (LPS). We characterized OSP-specific immune responses in adult recipients of an oral killed cholera vaccine (OCV WC-rBS) and compared these with responses in patients with cholera caused by Vibrio cholerae O1 Ogawa. Although vaccinees developed plasma immunoglobulin G (IgG), IgM, IgA antibody and antibody secreting cell (ASC, marker of mucosal response) to Ogawa OSP and LPS 7 days after vaccination, responses were significantly lower than that which occurred after cholera. Similarly, patients recovering from cholera had detectable IgA, IgM, and IgG memory B cell (MBC) responses against OSP and LPS on Day 30 and Day 90, whereas vaccinees only developed IgG responses to OSP 30 days after the second immunization. The markedly lower ASC and MBC responses to OSP and LPS observed among vaccinees might explain, in part, the lower protection of an OCV compared with natural infection.

The hierarchical process of differentiation of long-lived antibody-secreting cells is dependent on integrated signals derived from antigen and IL-17A.

Switched CD19-positive memory B cells purified from mice with chronic immune response against Thalassophrynenattereri venom proteins were cultured with venom or cytokines. Our results confirm the existence of a hierarchic process of differentiation: activated memory B cells progressively acquire increasing levels of CD138 and decreasing levels of CD45R/B220 to finally arrive at ASC with B220(neg) phenotype, which are IgG1-secreting cells. Only Bmem from peritoneal cavity or bone marrow of VTn immunized mice presented the capacity to generate ASC functionally active. IL-17A or IL-21/IL-23/IL-33 improves the ability of venom to induce intracellular IgG of peritoneal derived-ASC. Cognate stimulation with venom and IL-17A is sufficient to down-regulate the expression of CD45R/B220. BAFF-R is up-regulated in splenic or medullar derived-ASC stimulated by venom, CpG or cytokines. Only splenic derived-ASC up-regulate Bcl-2 expression after CpG or the combination of IL-21/IL-23/IL-33 stimulation. Finally, the activation of ASC for IgG1 secretion is triggered by venom proteins in peritoneal cavity and by IL-17A in medullar niche. These results show the importance of the integration of signals downstream of BCR and IL17-A receptors in modulating ASC differentiation, focusing in the microenvironment niche of their generation.