IgG Secretion Research Articles

Lymph node and circulating TFH cells support SIV-specific antibody responses in peripheral blood and genital tract of vaccinated female rhesus macaques

Abstract TFH cells are critical for Ag-specific B cell selection in germinal centers (GCs). Circulating blood CXCR5+ CD4+ T cells (cTFH) were implicated in development of broadly neutralizing antibodies in HIV+ patients. We asked if an SIV vaccine regimen could induce GC TFH and cTFH cells that supported anti-SIV humoral immunity. Female rhesus macaques (RM) were primed twice mucosally with replicating Ad5hr-SIV recombinants (wks0&13) and boosted twice intramuscularly with SIVgp120 proteins (wks26&38). Blood, lymph nodes (LN), bone marrow (BM), vaginal and rectal swabs were collected pre-vaccination, post-2nd prime and post-2nd boost for TFH and B cell assessment. Total TFH, and Env-specific GC TFH cells and Env-specific memory (ESM) B cells were seen at wk38+3d, suggesting vaccine-mediated GC maturation. Env-specific LN TFH cells and GC B cells at wk13+3d correlated respectively with Env-specific rectal IgA and vaginal IgG titers at wk16. GC B cells and ESM B cells at wk38+3d correlated with Env-specific vaginal IgG and rectal IgA titers at wk41 respectively, indicating early GC formation contributed to SIV-specific immunity in the female genital tract (FGT). Env-specific cTFH cells peaked post-boosting and positively correlated with Env-specific IgA and IgG secreting plasmablasts and plasma cells in BM and rectal IgA titers. cTFH cells induced CD38 and CD138 upregulation in autologous B cells and supported IgA secretion in cocultures compared to B cells cultured alone or with non-cTFH cells. Our data indicate the prime/boost regimen induced GC TFH and cTFH cells, enabling robust anti-SIV T-cell-dependent mucosal and systemic B cell responses. Both LN and circulating TFH cells may play pivotal roles in FGT protective humoral immunity.

In vivo mechanisms by which Irf5 regulates BCR-, TCR- and TLR-induced plasma cell generation and antibody secretion

Abstract Generation of antibody secreting cells (ASCs) requires multiple B cell activation pathways, including B cell receptor (BCR), T cell receptor (TCR), and Toll-like receptors (TLRs). In human naïve B cells, we reported that knockdown of interferon regulatory factor 5 (IRF5) resulted in IgD retention, reduced proliferation and plasma cell differentiation, and reduced IgG secretion. Defects were due to early impairments in B cell activation and clonal expansion. Conversely, murine Irf5 was reported to regulate antibody production through direct control of class switch recombination (CSR) at the γ2a locus. To further elucidate distinct and overlapping roles between human and murine IRF5 in the regulation of plasma cell differentiation and antibody production, we performed in vivo analysis of B and T cell differentiation and function in wild-type (wt) and Irf5 knockout (ko) littermate mice after immunization. B and T cell subsets were analyzed by multi-color flow cytometry. Naïve B cells were in vitro differentiated to ASCs with CpG-B, anti-IgM, anti-CD40 and IL-21. Kinetics of B cell activation, AID expression and IgG production were determined. BCR and TCR signaling were examined by phospho-flow. A combination of T-dependent/independent and TLR-dependent/independent immunizations were used to study ASC differentiation. RAG−/− mice were used to determine cell type-specific effects. Results indicate distinct roles for human and murine IRF5 at the early stages of BCR signaling and redundant roles at the later stages of CSR, plasma cell differentiation and antibody secretion via regulation of AID.

The relationship between the immune system activity and the bacterial vaginosis development

The main cause of bacterial vaginosis (BV) is immunodeficiency which is both systemic and local in nature and increases in parallel to a dysbiosis grade deepening. It is of great importance to link the indicators of BV-associated microbiota with individual indicators of systemic immune responses. The aim of the study is to establish a relationship between the immune system state and the development of various grades bacterial dysbiosis and BV. The study selected 298 women, who were divided into groups according to the index of conditionally pathogenic microflora (ICPM): in normocenosis ICPM was lower than -3 lg GE/sample (n = 53); in grade I dysbiosis it was from -3 to -1 lg GE/sample (n = 128); and in grade II dysbiosis (BV) it was more than -1 lg GE/sample (n = 117). Molecular-genetic studies of the scraping of epithelium from the posterolateral vaginal paries were performed using the method of polymerase chain reaction (DNA technology, RF). Optional and obligate anaerobes, myco- and ureplasms and yeast fungi were quantified. Content in the blood of fractions of T-and B-lymphocytes, immunoglobulins, cytokines, complement, lymphocyte phagocytic activity (LPA), 21 indicators in total) were determined using standard immunological methods. Interrelation between the microbial biocenosis with the considered indicators was studied using the regression analysis (Statistica 10; StatSoft, Inc.,USA).In normocenosis, normobiota indicator (NBI) was determined by activation of complement (C4), PA – as well by activation of complement (C3) and by content of sIgA in blood; ObA and YF – by γ-INF level, and YF – also by IL8, IL10 and sIgA levels; ICPM effective value was determined by the number of CD22+ lymphocytes. In grade I dysbiosis, NBI depended on the number of CD22+ lymphocytes, IL6, IgM, IgG, LPI, CIC, IL10, which indicated the implementation of various mechanisms and involvement of almost all of the immune system levers to compensate for dysbiosis at the beginning of its formation. In grade I dysbiosis, ObA amount was also affected by IL6 and IL10, and MU amount – by IL1β and IgG. In grade II dysbiosis, no factors affected NBI, which indicated that the dysbiotic processes went beyond the control of the immune system. Content of IL1β in blood reflected a shift of the ICPM: when its level exceeded 24.6 pg/ml, grade II dysbiosis was present; at IL1β level from 9.6 pg/ml to 24.6 pg/ml – it was grade I dysbiosis, and at IL1β level less than 9.6 pg/ml – it was normocenosis. Factors, which anaerobic dysbiosis was the trigger for, were activation of the humoral immunity (IgG secretion) and IL10 increase.General analysis of the interrelation between the immune system indicators with the number of pathogenic microbiota revealed the development of systemic combined immunodeficiency and “slipping” of the most pathogenic microorganisms out of the control of the immune system.

Oral uptake and persistence of the FnAb-8 protein characterized by in situ radio-labeling and PET/CT imaging

The absorption of peptides and proteins delivered orally is minimum because of the intestine epithelial barrier. There are few known active transport mechanisms for macromolecules including the neonatal Fc Receptor (FcRn) for the absorption and secretion of IgGs in infant and adult intestine. We had previously described the FnAb-8 protein that could bind to hFcRn tightly at pH 6.0 but barely at pH 7.4. In this study, we examined its uptake, biodistribution and pharmacokinetics after peroral administration in both wild-type and human FcRn transgenic (Tg) mice. FnAb-8 was modified to contain trans-cyclooctene (TCO) which could interact with 18F labeled tetrazine in situ via the bioorthogonal inverse-electron-demand Diels−Alder reaction. We showed that FnAb-8 had a tendency to distribute and persist in the Tg mice intestine for an extended duration of time. It could also be absorbed into the circulation and distributed systemically over a long period of time up to 172 h. The improvement in oral uptake and concentration in the intestine tissue may be valuable for designing oral delivery of biopharmaceuticals, especially for diseases involving the gastric intestinal tissue.

In Human Autoimmunity, a Substantial Component of the B Cell Repertoire Consists of Polyclonal, Barely Mutated IgG+ve B Cells.

B cells are critical for promoting autoimmunity and the success of B cell depletion therapy in rheumatoid arthritis (RA) confirms their importance in driving chronic inflammation. Whilst disease specific autoantibodies are useful diagnostically, our understanding of the pathogenic B cell repertoire remains unclear. Defining it would lead to novel insights and curative treatments. To address this, we have undertaken the largest study to date of over 150 RA patients, utilizing next generation sequencing (NGS) to analyze up to 200,000 BCR sequences per patient. The full-length antigen-binding variable region of the heavy chain (IgGHV) of the IgG B cell receptor (BCR) were sequenced. Surprisingly, RA patients do not express particular clonal expansions of B cells at diagnosis. Rather they express a polyclonal IgG repertoire with a significant increase in BCRs that have barely mutated away from the germline sequence. This pattern remains even after commencing disease modifying therapy. These hypomutated BCRs are expressed by TNF-alpha secreting IgG+veCD27−ve B cells, that are expanded in RA peripheral blood and enriched in the rheumatoid synovium. A similar B cell repertoire is expressed by patients with Sjögren's syndrome. A rate limiting step in the initiation of autoimmunity is the activation of B cells and this data reveals that a sizeable component of the human autoimmune B cell repertoire consists of polyclonal, hypomutated IgG+ve B cells, that may play a critical role in driving chronic inflammation.

B cell-derived anti-beta 2 glycoprotein I antibody mediates hyperhomocysteinemia-aggravated abdominal aortic aneurysm

Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysm (AAA). Hyperhomocysteinemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibody that ultimately contributes to AAA formation. ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of lipid metabolites in B cells by LC-MS/MS, which contributed to elevated anti-β2GPI IgG secretion. By use of the Toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that the culture supernatant of Hcy-activated B cells and HHcy plasma IgG independently polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- or CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG for HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice, then transferred these IgG into μMT mice and subsequently subjected them to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG had significantly exacerbated elastase- or CaPO4-induced AAA development accompanied with increased elastin degradation, MMP2/9 expression and anti-β2GPI IgG deposition in vascular lesions by immunofluorescence histochemical staining. Thus, HHcy aggravates AAA formation by activating B cells to secret anti-β2GPI IgG.

Correction of autophagy impairment inhibits pathology in the NOD.H-2h4 mouse model of primary Sjögren's syndrome

Dysregulation of autophagy has been implicated in the development of various disease indications including autoimmune diseases. Here we identified hitherto unsuspected molecular alterations of autophagy occurring at an early stage of the macroautophagy pathway in the salivary glands and spleen of NOD.H-2h4 mice that develop a primary Sjögren's-like syndrome. In this study we investigated the capacity of phosphopeptide P140 to correct immune alteration in NOD.H-2h4 mice and the effect on neogenesis of tertiary lymphoid structures in salivary glands, which is hallmark characteristic of SS. Phosphopeptide P140 known to lower excessive autophagy processes, rescued sick NOD.H-2h4 mice from some autophagy defects and significantly reduced formation of tertiary lymphoid structures in salivary glands. Mechanistically, the frequency of activated CD44high/CD62Llow CD4+ T cell populations was significantly decreased and this reduction was correlated with an increased number of CD44low/CD62Lhigh resting T cells. The CD8 T cell compartment was not affected. P140 down-regulated the maturation and differentiation of B cells into plasma cells, and decreased IgG and autoantibody secretion. It had no effect on germinal centers B cells (B220+ FAS+GL-7+) that are an important compound of the B cell humoral immune response. Together with previous data generated in MRL/lpr mice that develop some features of Sjögren's syndrome associated to other inflammatory and autoimmune defects, our present findings strongly reinforce the potential of autophagy modulators, such as P140, for treating patients with Sjögren's syndrome.

Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73–1.0), specificity of 0.66 (95% CI 0.52–0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75–0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47–0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Self-Antigens Displayed on Liposomal Nanoparticles above a Threshold of Epitope Density Elicit Class-Switched Autoreactive Antibodies Independent of T Cell Help.

Epitope density has a profound impact on B cell responses to particulate Ags, the molecular mechanisms of which remain to be explored. To dissect the role of epitope density in this process, we have synthesized a series of liposomal particles, similar to the size of viruses, that display a model self-antigen peptide at defined surface densities. Immunization of C57BL/6J mice using these particles elicited both IgM and class-switched IgG1, IgG2b, and IgG3 autoreactive Abs that depended on the epitope density. In C57BL/6 gene knockout mice lacking either functional TCRs or MHC class II molecules on B cells, the liposomal particles also elicited IgM, IgG1, IgG2b, and IgG3 responses that were comparable in magnitudes to wild-type mice, suggesting that this B cell response was independent of cognate T cell help. Notably, the titer of the IgG in wild-type animals could be increased by more than 200-fold upon replacement of liposomes with bacteriophage Qβ virus-like particles that displayed the same self-antigen peptide at comparable epitope densities. This enhancement was lost almost completely in gene knockout mice lacking either TCRs or MHC class II molecules on B cells. In conclusion, epitope density above a threshold on particulate Ags can serve as a stand-alone signal to trigger secretion of autoreactive and class-switched IgG in vivo in the absence of cognate T cell help or any adjuvants. The extraordinary immunogenicity of Qβ viral-like particles relies, in large part, on their ability to effectively recruit T cell help after B cell activation.

Investigating the role of ERAD on antibody processing in glycoengineered Saccharomyces cerevisiae.

N-glycosylation plays an important role in the endoplasmic reticulum quality control (ERQC). N-glycan biosynthesis pathways have been engineered in yeasts and fungi to enable the production of therapeutic glycoproteins with human-compatible N-glycosylation, and some glycoengineering approaches alter the synthesis of the lipid-linked oligosaccharide (LLO). Because the effects of LLO engineering on ERQC are currently unknown, we characterized intracellular processing of IgG in glycoengineered Δalg3 Δalg11 Saccharomyces cerevisiae strain and analyzed how altered LLO structures affect endoplasmic reticulum-associated degradation (ERAD). Intracellular IgG light and heavy chain molecules expressed in Δalg3 Δalg11 strain are ERAD substrates and targeted to ERAD independently of Yos9p and Htm1p, whereas in the presence of ALG3 ERAD targeting is dependent on Yos9p but does not require Htm1p. Blocking of ERAD accumulated ER and post-Golgi forms of IgG and increased glycosylation of matα secretion signal but did not improve IgG secretion. Our results show ERAD targeting of a heterologous glycoprotein in yeast, and suggest that proteins in the ER can be targeted to ERAD via other mechanisms than the Htm1p-Yos9p-dependent route when the LLO biosynthesis is altered.

Toolkit for the study of yellow fever virus

Yellow fever virus (YFV) is a dangerous re-emerging mosquito borne flavivirus with a high lethality, which causes untreatable haemorrhagic fever in humans. Important disadvantages regarding safety and limited vaccine supply have been identified in the existing live attenuated vaccine. Alternative safer therapeutic strategies are needed to reduce the lethality of infections and protect a wider group of people. To this point, we created an array of essential tools for the study of YFV. We designed a novel YFV reporter virus to develop a highly sensitive high throughput neutralisation assay (Z’=0.65), which can screen virus inhibitors in only 48 hours. Moreover, we designed a site-specific biotinylated soluble YF envelope (E) protein by a cloning strategy involving the use of a biotin acceptor peptide and E. coli Biotin-protein birA ligase. The biotinylated E protein was used to develop a highly sensitive ELISA to screen antibodies that bind to the YFV E protein. Furthermore, we immunised mice with a soluble YFV E protein and generated anti-YFV E IgG secreting hybridoma cell lines. Monoclonal antibodies secreted by hybridomas were purified by affinity chromatography and are being characterised. The developed antibodies against YF E protein are a crucial tool for the molecular study of YF. Additionally, neutralising antibodies against YFV could potentially be developed into the first therapeutic treatment against YF infection. The toolkit developed in this project includes a neutralisation assay, a binding ELISA, and anti YFV E antibodies. These are essential instruments to expand the knowledge of YFV.

B cell-derived anti-beta 2 glycoprotein I antibody contributes to hyperhomocysteinaemia-aggravated abdominal aortic aneurysm.

Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation. ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-β2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into μMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-β2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining. Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-β2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

Complexin 2 regulates secretion of immunoglobulin in antibody-secreting cells.

IntroductionComplexins (CPLXs), initially identified in neuronal presynaptic terminals, are cytoplasmic proteins that interact with the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis, their distribution and role in immune cells are still unclear. In this study, we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes.MethodsWe examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo, we analyzed the immune phenotype of CPLX2 KO mice. Furthermore, antibodies secretion from the peritoneal cavity, spleen, and bone marrow cells of wild‐type (WT) and CPLX2 KO mice were determined.ResultsCPLX2 was detected in B cells but not in T cells, while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes, serum concentrations of immunoglobulin G (IgG), IgA, IgM, and IgE were measured in WT and CPLX2 KO mice using enzyme‐linked immunosorbent assay. The level of IgM, which mainly consists of natural antibodies, was higher in KO mice than that in WT mice, while the levels of other antibodies were similar in both types of mice. Additionally, we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody‐secreting cells (ASCs) of CPLX2 KO mice.ConclusionOur data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs.

Exploiting the Lymph-Node-Amplifying Effect for Potent Systemic and Gastrointestinal Immune Responses via Polymer/Lipid Nanoparticles.

Parenteral vaccinations are not able to elicit effective systemic and gastrointestinal immune protection simultaneously because the lymphocytes are typically restricted to primed tissues. Although all-trans retinoic acid (atRA) was reported to trigger the gut-homing of immunocytes, the bioavailability and systemic immune responses remain limited for use in robust enteric vaccinations. Here, we show that co-delivery of atRA, CpG oligodeoxynucleotides (CpG), and antigens via engineered polymer/lipid nanoparticles (PLNPs) could exploit the amplifying function of draining lymph nodes (DLNs) for potent gut tropism and immune activations. After intramuscular injection, forming an immune-potentiated environment at the injection site, the PLNPs induced the designated transfer of primed dendritic cells (DCs) to the DLNs instead of the gastrointestinal tissues. Within the DLNs, the immune-potentiated environment markedly amplified the antigen presentation and homing receptor switch among immunocytes, which simultaneously stimulated the preferential dissipation of activated lymphocytes in the peripheral and gastrointestinal tissues, that is, exerted a DLN-amplifying effect. Compared with current atRA-containing formulations, the PLNPs not only boosted potent IgG secretions and T cell activations in the peripheral tissue but also provoked robust T cell homing and antigen-specific IgA levels in the gastrointestinal tracts in both ovalbumin and EV71 vaccinations. These data indicate that exploiting DLN amplification can stimulate potent systemic and gastrointestinal responses for more efficient enteric vaccinations.

T‐cell–derived extracellular vesicles regulate B‐cell IgG production via pyruvate kinase muscle isozyme 2

Intercellular communication between lymphocytes plays a fundamental role in numerous immune responses. Previously, we demonstrated that hyperhomocysteinemia (HHcy) induced T cell intracellular glycolytic-lipogenic reprogramming and IFN-γ secretion via pyruvate kinase muscle isozyme 2 (PKM2) to accelerate atherosclerosis. Usually, B cells partially obtain help from T cells in antibody responses. However, whether PKM2 activation in T cells regulates B cell antibody production is unknown. Extracellular vesicles (EVs) are important cellular communication vehicles. Here, we found that PKM2 activator TEPP46-stimulated T-cell-derived EVs promoted B-cell IgG secretion. Conversely, EVs secreted from PKM2-null T cells were internalized into B cells and markedly inhibited B-cell mitochondrial programming, activation, and IgG production. Mechanistically, lipidomics analyses showed that increased ceramides in PKM2-activated T-cell EVs were mainly responsible for enhanced B cell IgG secretion induced by these EVs. Finally, quantum dots (QDs) were packaged with PKM2-null T cell EVs and anti-CD19 antibody to exert B-cell targeting and inhibit IgG production, eventually ameliorating HHcy-accelerated atherosclerosis in vivo. Thus, PKM2-mediated EV ceramides in T cells may be an important cargo for T-cell-regulated B cell IgG production, and QD-CD19-PKM2-null T cell EVs hold high potential to treat B cell overactivation-related diseases.-Yang, J., Dang, G., Lü, S., Liu, H., Ma, X., Han, L., Deng, J., Miao, Y., Li, X., Shao, F., Jiang, C., Xu, Q., Wang, X., Feng, J. T-cell-derived extracellular vesicles regulate B-cell IgG production via pyruvate kinase muscle isozyme 2.

Development of an ic-ELISA and immunochromatographic strip based on IgG antibody for detection of ω-conotoxin MVIIA.

ω-conotoxin MVIIA(ω-CTX MVIIA) is a peptide consisting of 25 amino acid residues secreted mainly by Conus magus. In view of the toxin threat to humans and animals and defined application in analgesic therapy, it is necessary to develop a rapid, effective and accuracy method for the quantification and analysis of ω-CTX MVIIA in real samples. In the present study, a hybridoma cell named 2E5 stable secreting IgG antibody against ω-CTX MVIIA was selected successfully, and the subtype of Mab 2E5 was IgG1. The purified monoclonal antibody(Mab) 2E5 has high affinity (about 2.79 × 109 L/mol), and shows high specificity to ω-CTX MVIIA antigen. The linear range of ic-ELISA to detect ω-CTX MVIIA was 0.20˜7.22 μg/mL, with a lower detection limit (LOD) of 0.14 ng/mL. The average recovery of intra- and inter-assay were (85.45 ± 2.28)% and (88.03 ± 4.80)% respectively, with a coefficient of variation from 2.59% to 5.42%. The LOD of colloidal strip by naked eye was 1 μg/mL, and the detection time was less than 10 min without any equipment. The developed ELISA and colloidal test strips based on this IgG antibody could be used to detect ω-CTX MVIIA residue in real Conus samples.

Human Colorectal Carcinoma Infiltrating B Lymphocytes Are Active Secretors of the Immunoglobulin Isotypes A, G, and M.

Despite the importance of tumor infiltrating B cells (TiBc) in immunological circuits, their functional role is scarcely investigated. Here, we analyzed immunoglobulin (Ig) secretion of the subtypes IgA, IgG, and IgM of TiBc from freshly resected primary and secondary colorectal carcinomas (CRC) by FluoroSpot (n = 30 CRC) directly ex vivo. High, intermediate, and low secretion was observed in 33%, 37%, and 30% of the tumors for IgA; in 10%, 27%, and 63% for IgG; and in 21%, 36%, and 50% for IgM, respectively. These ex vivo data validate our previous findings: Most TiBc present in the CRC microenvironment are functional since they produce and actively secrete Ig (IgA > IgG > IgM). Of note, the presence of major histocompatibility complex (MHC) class II expressing cells in the tumor micromilieu only correlated with IgG secretion (p = 0.0004). Supporting recent findings in several other tumor entities, TiBc in CRC thus likely can contribute to tumor control in a dual role of sole antigen-presentation and additionally anti-tumoral Ig-production.

In vitro and in vivo cancer cell apoptosis triggered by competitive binding of Cinchona alkaloids to the RING domain of TRAF6.

TRAF6 is highly expressed in many tumors and plays an important role in the immune system. The aim of this study is to confirm anti-tumor activities of all naturally occurring Cinchona alkaloids that have been screened using computational docking program, and to validate the accuracy and specificity of the RING domain of TRAF6 as a potential anti-tumor target, and to explore their effect on the immune system. Results reported herein would demonstrate that Cinchona alkaloids could induce apoptosis in HeLa cells, inhibit the ubiquitination and phosphorylation of both AKT and TAK1, and up-regulate the ratio of Bax/Bcl-2. In addition, these compounds could induce apoptosis in vivo, and increase the secretion of TNF-α, IFN-γ, and IgG, while not significantly impacting the ratio of CD4+T/CD8+T. These investigations suggest that the RING domain of TRAF6 could serve as a de novo biological target for therapeutic treatment in cancers.

PB2152 THE ROLE OF THE BONE MARROW MICROENVIRONMENT AND CYTOKINES IN THE PROGRESSION OF MONOCLONAL GAMMAPATHY AND MULTIPLE MYELOMA IN PATIENTS OF THE GOMEL REGION OF BELARUS

Background:Multiple myeloma (MM) is an incurable tumor of hematopoietic tissue. In its development, it goes through a stage of monoclonal gammopathy (MGUS) or smoldering myeloma, when a tumor clone is born. With monoclonal gammopathy, the percentage of clonal plasma cells is less than 10%, therefore, during cytological examination of the bone marrow, it is not always possible to determine any specific differences between myeloma cells and normal plasma, therefore, an immunohistochemical study of the bone marrow and a combination of various biochemical markers help in this situation.Aims:Assess the role of the bone marrow microenvironment and the prognostic significance of some cytokines in the progression of diseases in monoclonal gammopathy and multiple myeloma.MethodsThe study was carried out on samples of whole venous blood, aspirate and bone marrow biopsy material of 30 patients (12 patients with monoclonal gammapathy and 18 patients with multiple myeloma). The median age was 64 years for MGUS and 65 years for MM.All patients have undergone blood tests for cytokines, immunoglobulins, kappa and lambda light chains, cytological, immunophenotypic and immunohistochemical studies of the bone marrow. Results were evaluated at the time of diagnosis. The number of clonal plasma cells in the bone marrow in patients with MGUS averaged 3.5% (0.6‐11.2%), in patients with MM 42.2% (12.2‐72.6%). According to immunochemical variants, MM and MGUS are represented by secretion of IgG, IgA, IgM, secretion of kappa and lambda light chains. The level of CD138 positive cells, kappa, lambda chains, the myelokariocyte count, the presence of bone marrow fibrosis were assessed immunohistochemically.Results:When comparing immunohistochemical data for MGUS and MM at the time of diagnosis, a significant excess of the level of CD138 positive cells was found with a significant excess of the count of kappa and lambda light chains in blood serum and bone marrow in patients with MM. In patients with MGUS, the CD138 positive cell count during immunohistochemistry was significantly higher than in the bone marrow immunophenotypic study (p &lt;0.0001) and was accompanied by significant kappa or lambda chains expression in the peripheral blood and bone marrow, which may indicate a possible early generalization of the process and MGUS progression in MM. In all patients with MGUS and expressed expression of CD138 positive cells, the immunohistochemical study showed a significant excess of TNF (p &lt;0.001; r = 0.693), interleukin‐6, interleukin‐8, and were accompanied by lambda light chain secretion (p &lt;0.001; r = 0.698), which suggests the production of cytokines by tumor plasma cells. During follow‐up (6 months) in two MGUS patients with these characteristics, the disease transformed into MM and in one patient with IgM secretion into Waldenstrom macroglobulinemia. Fibrotic changes in the bone marrow during MGUS were combined with lambda light chain production, increased production of interleukin‐6 and interleukin‐8, a decrease in the number of megakaryocytes in the bone marrow (p &lt;0.001; r = 0.698), which may indicate a bone marrow reassociation associated with possible tumor progression.Summary/Conclusion:When analyzing the results of bone marrow immunohistochemical indicators, blood cytokines in our study revealed an increase in the number of CD138 positive cells and a significant excess of cytokines in the producing tumor cells in MGUS, the appearance of fibotic changes and a decrease in the number of myelokariocyte, which can be a significant biomarker of the risk of progression in monoclonal gammapathy.

Effects of Candida albicans infection on defense effector secretion by human oral mucosal epithelial cells

ObjectiveThe aim of this study was to investigate the effects of Candida albicans on the production of defense effector molecules by human oral mucosal epithelial cells in vitro. DesignImmortalized human oral mucosal epithelial (Leuk-1) cells and C. albicans strain 5314 were cocultured at different cell-to-C. albicans ratios. The viability of Leuk-1 cells was determined by MTT and RTCA measurements. The secretory levels of multiple defense effector molecules were determined by Enzyme-linked immunosorbent assay (ELISA). ResultsOur results indicated that C. albicans significantly decreased the secretion of IgG, cystatin C, lactoferrin, and TGF-β1 in a dose-dependent manner and remarkably reduced the production of IgA independent of the cell-to-C. albicans ratio. However, C. albicans clearly enhanced the secretion of IgM, galectin-3, P-selectin, granzyme B and perforin. ConclusionThese results suggest that C. albicans may exert a regulatory role in the defense response of oral mucosal epithelial cells by altering secretory levels of defense effector molecules.