Circulating pathogen-specific plasmablasts in female patients with upper genital tract infection.

Mucosal antibodies constitute the first line of adaptive immune defence against invaders in the female genital tract (FGT), yet the sequence of events leading to their production is surprisingly poorly characterized. We explored the induction of pathogen-specific antibody-secreting cells (ASC) as a response to an acute infection in the upper FGT. We recruited 12 patients undergoing surgery due to an upper FGT infection (7/12 blood culture positive, 5/12 negative) and six healthy controls. Pathogens were sampled during surgery and PBMC collected in the acute phase of the disease (days 7-10). We searched by ELISPOT circulating pathogen-specific ASC and explored their frequency, immunoglobulin isotype distribution, and expressions of homing receptors (α4ß7, L-selectin, and CLA). All patients had circulating ASC specific to the infective bacteria; the geometric mean was 434 (95%CI 155-1234) ASC (IgA + IgG + IgM)/106 PBMC. IgA ASC predominated in 7/12, IgG ASC in 3/12, and IgM ASC in 2/12 cases. Of all the pathogen-specific ASC, 60% expressed α4ß7, 67% L-selectin, and 9% CLA. This study is the first to show induction of pathogen-specific ASC in the peripheral blood in bacterial infection in the human FGT. Our findings reveal that such FGT-originating pathogen-specific ASC are predominated by IgA ASC and exhibit a homing receptor profile resembling that of ASC in acute urinary tract infection. The data thus suggest a characteristic profile shared by the urogenital tract.

IL-17 Promotes Differentiation of Splenic LSK- Lymphoid Progenitors into B Cells following Plasmodium yoelii Infection.

Lineage-Sca-1+c-Kit- (LSK-) cells are a lymphoid progenitor population that expands in the spleen and preferentially differentiates into mature B cells in response to Plasmodium yoelii infection in mice. Furthermore, LSK- derived B cells can subsequently contribute to the ongoing immune response through the generation of parasite-specific Ab-secreting cells, as well as germinal center and memory B cells. However, the factors that promote their differentiation into B cells in the spleen postinfection are not defined. In this article, we show that LSK- cells produce the cytokine IL-17 in response to Plasmodium infection. Using Il-17ra-/- mice, IL-17R signaling in cells other than LSK- cells was found to support their differentiation into B cells. Moreover, primary splenic stromal cells grown in the presence of IL-17 enhanced the production of CXCL12, a chemokine associated with B cell development in the bone marrow, by a population of IL-17RA-expressing podoplanin+CD31- stromal cells, a profile associated with fibroblastic reticular cells. Subsequent blockade of CXCL12 in vitro reduced differentiation of LSK- cells into B cells, supporting a direct role for this chemokine in this process. Immunofluorescence indicated that podoplanin+ stromal cells in the red pulp were the primary producers of CXCL12 after P. yoelii infection. Furthermore, podoplanin staining on stromal cells was more diffuse, and CXCL12 staining was dramatically reduced in Il-17ra-/- mice postinfection. Together, these results identify a distinct pathway that supports lymphoid development in the spleen during acute Plasmodium infection.

Early Emergence of CD19-Negative Human Antibody-Secreting Cells at the Plasmablast to Plasma Cell Transition.

Long-lived human plasma cells (PCs) play central roles in immunity and autoimmunity and are enriched among the subpopulation of CD19neg human PCs. However, whether human CD19neg PCs are necessarily aged cells that have gradually lost CD19 expression is not known. Assessing peripheral blood samples at steady-state and during the acute response to influenza vaccination in healthy donors, we identify the presence of phenotypic CD19neg plasmablasts, the proliferative precursor state to mature PCs, and demonstrate by ELISPOT that these are Ab-secreting cells (ASCs). During the acute response to influenza vaccination, CD19pos, CD19low, and CD19neg ASCs secrete vaccine-specific Abs and show linked IGHV repertoires. To address precursor/product relationships, we use in vitro models that mimic T-dependent and T-independent differentiation, finding that the CD19neg state can be established at the plasmablast to PC transition, that CD19neg PCs increase as a percentage of surviving PCs in vitro, and that CD19neg and CD19pos PCs can be maintained independently. These data provide proof-of-principle for the view that newly generated ASCs can acquire a mature PC phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation and that aging is not an obligate requirement for a CD19neg state to be established.

An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation.

BACKGROUND: CNS inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse B cell subsets. Although antibody secreting cells (ASC) within the inflamed CNS have been extensively examined, memory B cell (Bmem) characterization has been limited as they do not secrete antibody without stimulation. Moreover, unlike human Bmem, reliable surface markers for murine Bmem remain elusive. NEW METHOD: Using a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC. COMPARISON WITH EXISTING METHODS: Stimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial ASC loss and minimal Bmem to ASC conversion of CNS-derived cells. By varying stimulation duration, TLR activators, and culture supplements, we achieved optimal conversion by culturing cells with TLR7/8 agonist R848 in the presence of feeder cells for 2days. RESULTS: Flow cytometry markers CD38 and CD73 characterizing murine Bmem from lymphoid tissue showed more diverse expression patterns on corresponding CNS-derived B cell subsets. Using the optimized TLR7/8 stimulation protocol, we compared virus-specific IgG Bmem versus pre-existing ASC within the brain and spinal cord. Increasing Bmem frequencies during chronic infection mirrored kinetics of ASC. However, despite initially similar Bmem and ASC accumulation, Bmem prevailed in the brain, but were lower than ASC in the spinal cord during persistence. CONCLUSION: Simultaneous enumeration of antigen-specific Bmem and ASC using the Bmem assay optimized for CNS-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation.

CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype.

Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2, independently of isotype, identified MBC subsets with distinct functions upon rechallenge. CD80(+)PD-L2(+) MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely, CD80(-)PD-L2(-) MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence, the differentiation and regeneration of MBCs are compartmentalized.

De novo oligoclonal expansions of circulating plasmablasts in active and relapsing IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a poorly understood, multiorgan, chronic inflammatory disease characterized by tumefactive lesions, storiform fibrosis, obliterative phlebitis, and accumulation of IgG4-expressing plasma cells at disease sites. OBJECTIVE: The role of B cells and IgG4 antibodies in IgG4-RD pathogenesis is not well defined. We evaluated patients with IgG4-RD for activated B cells in both disease lesions and peripheral blood and investigated their role in disease pathogenesis. METHODS: B-cell populations from the peripheral blood of 84 patients with active IgG4-RD were analyzed by using flow cytometry. The repertoire of B-cell populations was analyzed in a subset of patients by using next-generation sequencing. Fourteen of these patients were longitudinally followed for 9 to 15 months after rituximab therapy. RESULTS: Numbers of CD19(+)CD27(+)CD20(-)CD38(hi) plasmablasts, which are largely IgG4(+), are increased in patients with active IgG4-RD. These expanded plasmablasts are oligoclonal and exhibit extensive somatic hypermutation, and their numbers decrease after rituximab-mediated B-cell depletion therapy; this loss correlates with disease remission. A subset of patients relapse after rituximab therapy, and circulating plasmablasts that re-emerge in these subjects are clonally distinct and exhibit enhanced somatic hypermutation. Cloning and expression of immunoglobulin heavy and light chain genes from expanded plasmablasts at the peak of disease reveals that disease-associated IgG4 antibodies are self-reactive. CONCLUSIONS: Clonally expanded CD19(+)CD27(+)CD20(-)CD38(hi) plasmablasts are a hallmark of active IgG4-RD. Enhanced somatic mutation in activated B cells and plasmablasts and emergence of distinct plasmablast clones on relapse indicate that the disease pathogenesis is linked to de novo recruitment of naive B cells into T cell-dependent responses by CD4(+) T cells, likely driving a self-reactive disease process.

TLR2, TLR4 and the MyD88 signaling are crucial for the in vivo generation and the longevity of long-lived antibody-secreting cells.

This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220(pos). TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220(pos) (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220(neg) in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220(pos) (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220(pos) (MyD88-independent) and ASC B220(neg) into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220(neg) in peritoneum and BM. Terminally differentiated ASC B220(neg) required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220(pos) rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220(pos) and ASC B220(neg).

Astrocyte-derived CXCL10 drives accumulation of antibody-secreting cells in the central nervous system during viral encephalomyelitis.

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10(-/-) but not CXCL9(-/-) mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10(-/-) mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9(-/-) mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9(-/-) or CXCL10(-/-) mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.

Analysis of complement and plasma cells in the brain of patients with anti-NMDAR encephalitis.

OBJECTIVES: Most patients with anti-NMDA receptor (NMDAR) encephalitis have intrathecal synthesis of antibodies, which cause a decrease of cell surface and synaptic NMDAR. Antibodies are immunoglobulin G (IgG)1 and IgG3 subtypes and can potentially activate complement. We examined whether complement immunoreactivity and antibody-secreting cells (plasma cells/plasmablasts) are present in the brain of these patients. METHODS: Cultured rat hippocampal neurons were used in an immunocytochemical assay to test whether patients' antibodies can fix complement. Using the same reagents (antibodies to C9neo, C(5b-9), C3), complement immunoreactivity was determined in the brain of 5 patients, the teratoma of 21 patients, and appropriate control tissues. A set of markers for B (CD20), T (CD3, CD4, CD8) and antibody-secreting cells (plasma cells/plasmablasts, CD138) were used to examine the brain inflammatory infiltrates. RESULTS: Patients' antibodies were able to bind complement in vitro, but deposits of complement were not detected in patients' brain. Parallel experiments with teratomas showed that in contrast to the brain, the neural tissue of the tumors contained complement. Analysis of the inflammatory infiltrates in brain samples from autopsy or biopsy performed 3-4 weeks after symptom presentation demonstrated numerous antibody-secreting cells (CD138+) in perivascular, interstitial, and Virchow-Robin spaces, and B and T cells predominantly located in perivascular regions. CONCLUSIONS: Complement-mediated mechanisms do not appear to play a substantial pathogenic role in anti-NMDAR encephalitis. In contrast, there are copious infiltrates of antibody-secreting cells (plasma cells/plasmablasts) in the CNS of these patients. The demonstration of these cells provides an explanation for the intrathecal synthesis of antibodies and has implications for treatment.

Sublingual immunotherapy mechanisms of action: the role of Th1 response.

Specific immunotherapy (SIT) is the only treatment able to modify the natural history of allergic subjects. Several aspects of the immunopathological response modified by sublingual immunotherapy (SLIT), which is an alternative route of administration for SIT, have been investigated. A shift from Th2-polarized immune response toward Th1-oriented pattern has been reported after SLIT. More recently, a crucial role for a subpopulation of T cells has been evidenced: T regulatory cells (Treg). Allergic patients have a defect of Tregs, and SLIT should be able to induce a specific Treg response. This issue is very relevant as the Treg-dependent cytokines, namely IL-10 and TGF-beta, are involved in the regulation of IgG and IgA antibodies production. Recent evidence shows that SLIT is also able of inducing a Treg response as detected by IL-10 production. IFNgamma is a typical Th1-dependent cytokine. SLIT may induce a significantly increased production of this cytokine and it may be considered as an early marker of SLIT response. Therefore, also SLIT is able of exerting the effects on immune response as well as the subcutaneous route.

Chemotherapy induced transient B-cell depletion boosts antibody-forming cells expansion driven by an epidermal growth factor-based cancer vaccine.

Cancer vaccines efficacy may improve inducing a rapid and persistent immune response, early at diagnosis along with standard therapies. EGF chemically conjugated to the carrier protein P64k from Neisseria meningitidis in montanide ISA 51 adjuvant is under evaluation, aiming to stimulate a B-cell response. High-dose cyclophosphamide and doxorubicin after priming enhanced the long-term frequency of EGF-specific antibody-forming cells (AFC) of IgM and IgG isotypes, but not the P64k response. Resulting combination, limitedly operational in Btk deficient xid mice, suggests that preferential B-cell lymphocyte space promoted by cyclophosphamide facilitates remaining EGF-specific AFC undergo homeostatic proliferation driven by boosting, amplifying the response.

Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR.

Truncation of the cytoplasmic tail of membrane-bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric epsilon-gamma1 BCR, consisting of the extracellular domains of the epsilon gene and the transmembrane and cytoplasmic domains of the gamma1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the "gamma1-mediated signalling" of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE-secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE-antibody secreting cells with "gamma1-signalling history" migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE-antibody secreting cells with WT "epsilon-signalling history". We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool than IgG1 plasmablasts.

Nature and functions of autoantibodies.

Antibodies that react with self-molecules occur in healthy individuals and are referred to as natural antibodies or autoantibodies. Natural autoantibodies are mainly IgM, are encoded by unmutated V(D)J genes and display a moderate affinity for self-antigens. They provide a first line of defense against infections, probably serve housekeeping functions and contribute to the homeostasis of the immune system. By contrast, high-affinity, somatically mutated IgG autoantibodies reflect a pathologic process whereby homeostatic pathways related to cell clearance, antigen-receptor signaling or cell effector functions are disturbed. In some autoimmune disorders, autoantibodies might be present before disease onset, show remarkable specificity and serve as biomarkers providing an opportunity for diagnosis and therapeutic intervention. In organ-specific autoimmune diseases, such as myasthenia gravis or pemphigus, autoantibodies directly bind to and injure target organs. In systemic autoimmune diseases, autoantibodies react with free molecules, such as phospholipids, as well as cell surface and nucleoprotein antigens, forming pathogenic antigen-antibody (immune) complexes. These autoantibodies injure tissues and organs through engagement of Fc gammaR activation of complement as well as internalization and activation of Toll-like receptors. Activation of intracellular Toll-like receptors in plasmacytoid dendritic cells leads to the production of type I interferon, whereas engagement of intracellular Toll-like receptors on antigen-presenting cells stimulates cell activation and the production of other inflammatory cytokines. Thus, immune complexes might perpetuate a positive feedback loop amplifying inflammatory responses.

Cutaneous antibody-secreting cells and B cells in a teleost fish.

Antibodies in cutaneous mucus and skin of teleosts play a critical role in the protective immune response against infection. We demonstrate by ELISPOT that antibody-secreting cells (ASC), which include LPS-inducible B cells (plasmablasts) and non-replicating plasma cells, reside in low numbers in the skin of channel catfish. Following immunization against the protozoan parasite Ichthyophthirius multifiliis, which infects skin and gills, the number of ASC in skin increased 20-fold, indicating that the number of ASC in skin is dynamic and increases in response to parasite infection. The number of ASC in skin remained elevated for at least 17 weeks after the last parasite exposure. Cutaneous ASC included I. multifiliis-specific ASC, which undoubtedly serve as the primary source of cutaneous antibodies that confer long-term humoral immunity against reinfection. Our demonstration that skin contains B cells and plasma cells suggests that it is an integral component of the teleost immune system.

Advances in antibody manufacturing using mammalian cells.

In this review, we describe recent advances in antibody processing technology including: (1) development of proprietary cell lines; (2) improved expression systems optimized by selective technologies to boost underperformers; (3) improved protein-free and serum-free culture media; and (4) attention to glycosylation and other post-translational modifications. Advances in computer technology and sophisticated redesign of bioreactors have been major contributors to the dramatic improvements in antibody yields that have been documented in the last decade. Disposable bioreactor components are now widespread, resulting in improved yields, better quality product and lower costs for producers. Downstream innovations include (1) disposable devices for clarification and purification, (2) improved resins and ligands, and (3) new designs of hardware for improved performance. While there are numerous factors contributing to the increased yields that have been obtained, the most sustained of these is the introduction of disposable technologies on both the upstream and the downstream ends of the process. With the continuing introduction of improved computer technology and technological innovation, there is every reason to believe that quality and quantity of antibody products will continue to improve in the coming years, and supply will be adequate to meet the forthcoming needs of the industry.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells.

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semi-quantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs.

Is long-term humoral immunity in the mucosa provided by long-lived plasma cells? A question still open.

The biology of antibody-secreting cells is still a matter of controversial discussion. One of the key questions is how the migration and lifespan of antibody-secreting cells is linked to their generation. A paper in this issue of the European Journal of Immunology provides further information on the generation of long-term mucosal immune responses; however, the study leaves many questions unanswered as discussed in this brief Commentary.

Antibody response of autogenous splenic tissue implanted in the abdominal cavity of mice.

There is still controversy about the immunologic function of autotransplanted splenic tissue. In this study, splenic autotransplantation was performed in the abdominal cavity of mice, and the plaque-forming cell (PFC) assay was used to investigate the frequency of antibody-forming cells in response to sheep red blood cell (SRBC) immunization. BALB/c mice were divided into four groups according to the location of the autogenous graft: intraomental (IO), free peritoneal splenosis (FPS), retroperitoneal (RP), and nongrafted control (CT). Thirty days after surgery the mice were immunized intraperitoneally with SRBCs, and 4 days later splenic immunoglobulin M anti-SRBC-secreting cells were determined by counting the number of PFCs. All the immunized mice showed increased numbers of PFCs that were about 2 logs higher than those in the the nonimmunized controls (P < 0.005). The frequencies of anti-SRBC-producing cells in the tissues grafted in various sites of the abdominal cavity (IO, FPS, RP), in the normal spleen from nonoperated controls (CT), or in the sham-operated control group (SCT) were not notably different (5582 +/- 2475 PFC/10(7) cells for IO; 4849 +/- 1856 for FPS; 6604 +/- 2903 for RP; 5940 +/- 5029 for CT; and 6172 +/- 2203 for SCT). Similar histology with small architectural variations was observed in all implants; less white pulp was involved, and there was more congestion in the red pulp, with extensive sinusoids and reticular fiber proliferation. This study shows that the T cell-dependent antibody response in implanted splenic tissues is as efficient as in the intact spleen, with no difference between the graft sites studied. This immune response does not depend on the slight architectural variations observed in the splenic implants.