Humoral Arm Research Articles

The Impact of Pentraxin 3 on Crohn's Disease Phenotype.

Pentraxin 3 [PTX3] is an acute-phase protein playing an important role in the regulation of the humoral arm of immune response. As one of the molecules from the conservative family of pentraxins, PTX3 is a soluble mediator involved in the transduction of pro-inflammatory signals between immunocompetent cells. Additionally, recognizing damage-associated molecular patterns (DAMPs) during tissue injury mediates wound healing; therefore, its concentration potentially correlates with the severity of fibrosis. The aim of our study was to evaluate the value of the PTX3 measurement as a phenotypic marker of the stenotic form of Crohn's disease. The research covered 63 patients, 35 with the narrowing type (B2) and 28 with the inflammatory type (B2) of CD. The mean concentrations of PTX3 in the study were as follows: 3.06 ng/mL (95% CI: 1.27-6.99) for the B1 phenotype, 4.89 ng/mL (95% CI: 2.98-13.65) for the B2 phenotype, and 3.04 ng/mL (95% CI: 1.01-4.97) for the control group. PTX3 concentrations reached the highest values in the B2 group and the lowest in the control group. The differences between the B1 and B2 groups were statistically significant at p < 0.001. The presented studies indicate the potential role of PTX3 in the monitoring of tissue remodeling and the development of fibrosis in CD.

Trivalent outer membrane vesicles-based combination vaccine candidate induces protective immunity against Campylobacter and invasive non-typhoidal Salmonella in adult mice.

Campylobacter and invasive non-typhoidal Salmonella (iNTS) are among the most common causative agents of gastroenteritis worldwide. As of now, no single combination licensed vaccine is available for public health use against both iNTS and Campylobacter species. Outer-membrane vesicles (OMVs) are nanoscale proteoliposomes released from the surface of gram-negative bacteria during log phase and harbor a variety of immunogenic proteins. Based on epidemiology of infections, we formulated a novel trivalent outer membrane vesicles (TOMVs)-based vaccine candidate against Campylobacter jejuni (CJ), Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE). Isolated OMVs from CJ, ST and SE were combined in equal ratios for formulation of TOMVs and 5µg of the developed vaccine candidate was used for intraperitoneal immunization of adult BALB/c mice. Immunization with TOMVs significantly activated both the humoral and cellular arm of adaptive immune response. Robust bactericidal effect was elicited by TOMVs immunized adult mice sera. TOMVs immunization induced long-term protective efficacy against CJ, ST and SE infections in mice. The study illustrates the ability of TOMVs-based combination immunogen in eliciting broad-spectrum protective immunity against prevalent Campylobacter and iNTS pathogens. According to the findings, TOMVs can work as a potent combination-based acellular vaccine candidate for amelioration of Campylobacter and iNTS-mediated gastroenteritis.

A novel multi-epitope peptide vaccine targeting immunogenic antigens of Ebola and monkeypox viruses with potential of immune responses provocation in silico.

The emergence or reemergence of monkeypox (Mpox) and Ebola virus (EBOV) agents causing zoonotic diseases remains a huge threat to human health. Our study aimed at designing a multi-epitope vaccine (MEV) candidate to target both the Mpox and EBOV agents using immunoinformatics tools. Viral protein sequences were retrieved, and potential nonallergenic, nontoxic, and antigenic epitopes were obtained. Next, cytotoxic and helper T-cell (CTL and HTL, respectively) and B-cell (BCL) epitopes were predicted, and those potential epitopes were fused utilizing proper linkers. The in silico cloning and expression processes were implemented using Escherichia coli K12. The immune responses were prognosticated using the C-ImmSim server. The MEV construct (29.53kDa) included four BCL, two CTL, and four HTL epitopes and adjuvant. The MEV traits were pertinent in terms of antigenicity, non-allergenicity, nontoxicity, physicochemical characters, and stability. The MEV candidate was also highly expressed in E. coli K12. The strong affinity of MEV-TLR3 was confirmed using molecular docking and molecular dynamics simulation analyses. Immune simulation analyses unraveled durable activation and responses of cellular and humoral arms alongside innate immune responses. The designed MEV candidate demonstrated appropriate traits and was promising in the prediction of immune responses against both Mpox and EBOV agents. Further experimental assessments of the MEV are required to verify its efficacy.

Features of T-cell and humoral immunity of patients with acute coronary syndrome, with and without COVID-19, depending on/peripheral blood B-lymphocytes with the phenotype CD3–CD19+CD5+ level

The aim of the work was to assess the T-cell and humoral immune arms in patients with acute coronary syndrome (ACS) recovered from or not exposed to COVID-19. 86 men with ACS aged 40 to 65 years old, who suffered or not exposed to COVID-19, which required stenting of coronary arteries, were examined. Depending on the peripheral blood B-lymphocyte CD3–CD19+CD5+ level and the former COVID-19 history, all patients were divided into 6 groups. Of the previously COVID-19 exposed subjects CD3–CD19+CD5+ lymphocytes were at: reduced (group 1), normal (group 2), increased (group 3) level. COVID-19 unexposed subjects had CD3–CD19+CD5+ lymphocyte level: reduced (group 4), normal (group 5), increased (group 6). The most severe clinical manifestations were observed in group 1: with a larger rate of stent thrombosis and increased mortality. In absolute numbers, T-lymphocytes were significantly lower in group 1 compared to other groups, except group 4. The lowest both relative and absolute T-helper cell counts were recorded in covid-19 patients, with reduced CD3–CD19+CD5+ cell level. NK-lymphocytes both in relative and absolute level were significantly (p 0.05) higher in patients who suffered from COVID-19 as compared to those in patients unexposed to previous COVID-19. Percentage of late-activation T-regulatory cells was minimal in patients with former COVID-19 along with high B-lymphocyte CD3–CD19+CD5+ level, which significantly (p 0.05) differed from those in subjects lacking former COVID-19 history. In addition, significantly lower B-lymphocyte CD3–CD19+CD5– level was found in group 1 and 4. The IgM level peaked in group 5 — subjects lacking former COVID-19 history and having normal CD3–CD19+CD5+ cell level that significantly differed in comparison with that of in group 1, 2, 3. At the same time, the maximum value of IgG level was recorded in COVID-19 patients with normal CD3–CD19+CD5+ cells, and significantly differed from those found in group 5 and 6. C1-inhibitor level was higher in group 3 that significantly differed from that in other groups. SARS-CoV-2-specific IgG and IgM levels were significantly higher in COVID-19-positive vs. COVID-19-negative patients. Conclusion. In patients who have suffered COVID-19 with low B-lymphocyte CD3–CD19+CD5+ level, a significantly compromised immune protection was noted in comparison with other groups: a decline in total T-lymphocyte, T-helper, early- and late-activation T-lymphocyte, T-regulatory cell, B-lymphocyte CD3–CD19+CD5– levels, which was associated with a more severe disease course characterized by stent thrombosis and large mortality.

Bhlhe40 Promotes CD4+ T Helper 1 Cell and Suppresses T Follicular Helper Cell Differentiation during Viral Infection.

In response to acute infection, naive CD4+ T cells primarily differentiate into T helper 1 (Th1) or T follicular helper (Tfh) cells that play critical roles in orchestrating cellular or humoral arms of immunity, respectively. However, despite the well established role of T-bet and BCL-6 in driving Th1 and Tfh cell lineage commitment, respectively, whether additional transcriptional circuits also underlie the fate bifurcation of Th1 and Tfh cell subsets is not fully understood. In this article, we study how the transcriptional regulator Bhlhe40 dictates the Th1/Tfh differentiation axis in mice. CD4+ T cell-specific deletion of Bhlhe40 abrogates Th1 but augments Tfh differentiation. We also assessed an increase in germinal center B cells and Ab production, suggesting that deletion of Bhlhe40 in CD4+ T cells not only alters Tfh differentiation but also their capacity to provide help to B cells. To identify molecular mechanisms by which Bhlhe40 regulates Th1 versus Tfh lineage choice, we first performed epigenetic profiling in the virus specific Th1 and Tfh cells following LCMV infection, which revealed distinct promoter and enhancer activities between the two helper cell lineages. Furthermore, we identified that Bhlhe40 directly binds to cis-regulatory elements of Th1-related genes such as Tbx21 and Cxcr6 to activate their expression while simultaneously binding to regions of Tfh-related genes such as Bcl6 and Cxcr5 to repress their expression. Collectively, our data suggest that Bhlhe40 functions as a transcription activator to promote Th1 cell differentiation and a transcription repressor to suppress Tfh cell differentiation.

Respiratory Syncytial Virus Vaccines: Analysis of Pre-Marketing Clinical Trials for Immunogenicity in the Population over 50 Years of Age.

Immunosenescence refers to age-related alterations in immune system function affecting both the humoral and cellular arm of immunity. Understanding immunosenescence and its impact on the vaccination of older adults is essential since primary vaccine responses in older individuals can fail to generate complete protection, especially vaccines targeting infections with increased incidence among the elderly, such as the respiratory syncytial virus. Here, we review clinical trials of both candidate and approved vaccines against respiratory syncytial virus (RSV) that include adults aged ≥50 years, with an emphasis on the evaluation of immunogenicity parameters. Currently, there are 10 vaccine candidates and 2 vaccines approved for the prevention of RSV in the older adult population. The number of registered clinical trials for this age group amounts to 42. Our preliminary evaluation of published results and interim analyses of RSV vaccine clinical trials indicates efficacy in older adult participants, demonstrating immunity levels that closely resemble those of younger adult participants.

IImmunological parameters in patients with chronic opisthorchiasis bearing gene mutations associated with osteoporosis-predisposition

To identify the features of the immune system functioning in patients with chronic opisthorchiasis bearing mutations in loci associated with predisposition to developing osteoporosis, comprehensive studies of cellular and humoral arms were carried out. The state of the phagocytic system was assessed by assessing absorption, metabolic activity and reactive oxygen species formation to restore nitrosine tetrazolium (spontaneous and stimulated NBT test). The phenotype of lymphocytes was determined by flow cytometry. The humoral immune arm was evaluated by the number of immunoglobulin classes M, G, A and E. Differences in the functional state of various arms of the immune system were revealed. In patients with chronic opisthorchiasis in the presence of rs1544410 polymorphism of the gene encoding the intracellular vitamin D receptor, the relative number of T-helper cells is significantly lower than in the group with the normal allele. In the presence of rs1800012 polymorphism of the gene encoding the α1-chain of type I collagen, the absolute lymphocyte count is significantly higher, spontaneous and stimulated NBT test were lower, the number of DN-T lymphocytes is significantly lower (both in relative and absolute values). In the presence of the rs3736228 mutation of the gene encoding the transmembrane low-density lipoprotein receptor, the level of myeloperoxidase and the neutrophil stimulation index are lower, the absorption activity of neutrophils is higher. The presence of the rs2234693 mutation for estrogen receptor gene leads to significantly increased level of stimulated NBT test and IgG concentration. Thus, patients with chronic opisthorchiasis bearing mutations in the COL1 A1, LRP5, ESR1(rs2234693) genes, have altered both nonspecific innate reactions and parameters of the adaptive immune arm; mutation of the VDR gene solely affects adaptive immunity. Analysis of the results suggests that the presence of mutations associated with the development of osteoporosis has a modulating effect on the immune response in chronic opisthorchiasis invasion. The identification of polymorphic genes associated with metabolic disorders of bone tissue and the study of the immunological profile in patients with chronic opisthorchiasis invasion will allow to implement an individual approach in the treatment of such patients.

Optimization and validation of flow cytometry method for quantification of SARS-CoV-2 antigen-reactive human memory T cells

A proper and representative monitoring of SARS-CoV-2 herd immunity including a long-term health impact on recovered patients and vaccinated individuals is of great importance. For this, a monitoring campaign should assesses both humoral and T-cell immune arms. Upon that, analyzing antigen specific-cell activation and cellular phenotype are informative. We developed a flow cytometry method for detection of intracellular IFN-producing antigen-reactive T cells after exposure of human peripheral blood mononuclear cells (PBMC) to SARS-CoV-2 virus antigens. The method was validated according to the following characteristics: sensitivity, specificity, precision, and robustness. We used positive samples from donors recovered from COVID-19 and negative samples from donors who had no contact with COVID-19 patients and lacking antibodies to SARS-CoV-2. All samples were tested by laboratory methods. Peripheral blood mononuclear cells were isolated from donor blood by centrifugation in a Ficoll density gradient. Specific T cells were stimulated with S-protein as well as N, M, ORF3a, and ORF7a protein peptides to count IFN-producing T cells by flow cytometer. The data were statistically analyzed. The area limited by ROC-curve and false positive rate (AUC) for CD4 and CD8 cells was from 0.97 to 1.00. Precision was considered acceptable because the coefficient of variation for all PBCM did not exceed 20%. Robustness was confirmed for frozen and freshly prepared PBMC samples. The thresholds levels to recognize immune and non-immune samples were defined for CD4-positive T-cells (0.029%) and CD8-positive T-cells (0.0640.068%). Also, acceptance criteria for positive and negative controls were defined. Based on the validation, the suitability of the method Evaluation of antigen-reactive T cells that produce intracellular IFN in response to SARS-CoV-2 virus antigens by flow cytometry was confirmed. The method allows for reliable data that was used to characterize standard control samples for internal quality control of TigraTest SARS-CoV-2 kits.

Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study

Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. OrganX and MSD Avenir.

Humoral Immunity Against Aspergillus fumigatus.

Aspergillus fumigatus is one the most ubiquitous airborne opportunistic human fungal pathogens. Understanding its interaction with host immune system, composed of cellular and humoral arm, is essential to explain the pathobiology of aspergillosis disease spectrum. While cellular immunity has been well studied, humoral immunity has been poorly acknowledge, although it plays a crucial role in bridging the fungus and immune cells. In this review, we have summarized available data on major players of humoral immunity against A. fumigatus and discussed how they may help to identify at-risk individuals, be used as diagnostic tools or promote alternative therapeutic strategies. Remaining challenges are highlighted and leads are given to guide future research to better grasp the complexity of humoral immune interaction with A. fumigatus.

The Tumor-Specific Immune Landscape in HPV+ Head and Neck Cancer.

Human papillomaviruses (HPVs) are the causative agent of several anogenital cancers as well as head and neck cancers, with HPV+ head and neck squamous cell carcinoma (HNSCC) becoming a rapidly growing public health issue in the Western world. Due its viral etiology and potentially its subanatomical location, HPV+ HNSCC exhibits an immune microenvironment which is more inflamed and thus distinct from HPV-negative HNSCC. Notably, the antigenic landscape in most HPV+ HNSCC tumors extends beyond the classical HPV oncoproteins E6/7 and is extensively targeted by both the humoral and cellular arms of the adaptive immune system. Here, we provide a comprehensive overview of HPV-specific immune responses in patients with HPV+ HNSCC. We highlight the localization, antigen specificity, and differentiation states of humoral and cellular immune responses, and discuss their similarities and differences. Finally, we review currently pursued immunotherapeutic treatment modalities that attempt to harness HPV-specific immune responses for improving clinical outcomes in patients with HPV+ HNSCC.

Design of a novel multi-epitope vaccine candidate against Chlamydia trachomatis using structural and nonstructural proteins: an immunoinformatics study

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium which causes eye and sexually transmitted infections. During pregnancy, the bacterium is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to infertility. The aim of our study was design of a multi-epitope vaccine (MEV) candidate against C. trachomatis. After protein sequence adoption from the NCBI, potential epitopes toxicity, antigenicity, allergenicity, MHC-I and MHC-II binding, cytotoxic T lymphocytes (CTLs), Helper T lymphocytes (HTLs) and interferon-γ (IFN-γ)- induction were predicted. The adopted epitopes were fused together using appropriate linkers. In the next step, the MEV structural mapping and characterization, three-dimensional (3D) structure homology modeling and refinement were also performed. The MEV candidate interaction with the toll-like receptor 4 (TLR4) was also docked. The immune responses simulation was assessed using the C-IMMSIM server. Molecular dynamic (MD) simulation verified the structural stability of the TLR4-MEV complex. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach demonstrated the MEV high affinity of binding to the TLR4, MHC-I and MHC-II. The MEV construct was also stable and water soluble and had enough antigenicity and lacked allergenicity with stimulation of T cells and B cells and INF-γ release. The immune simulation confirmed acceptable responses of both the humoral and cellular arms. It is proposed that in vitro and in vivo studies are needed to evaluate the findings of this study. Communicated by Ramaswamy H. Sarma

Poststroke Lung Infection by Opportunistic Commensal Bacteria Is Not Mediated by Their Expansion in the Gut Microbiota.

Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection. Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments. Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae. Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection.

Cellular and humoral immune parameters in the peripheral and local blood flow in patients with primary pelvic varicose

Pelviс varicose veins (PVV) in women is of extremely high relevance due to a close relationship with reproductive disorders and disease relapse. Despite on research in the field, the results of the analysis on immune reactivity in PVV are rather contradictory, and mainly relate to general mechanisms. The study of cell-mediated and humoral arms of adaptive immunity will allow us to assess an intensity and dynamics of PVV progression as well as a potential for using immune status parameters to optimize diagnosis and immunological correction. The aim of this study was to assess characteristics of cellular and humoral immunity in peripheral (ulnar) and local (ovarian) venous pools in women with mild and moderate PVV. The study involved 142 women of reproductive age (mean age 37.27.1 years) with diagnosed PVV mild (Group 1) (n = 79) and moderate (Group 2) (n = 63) forms. Data from 30 apparently healthy women (mean age 33.56.3 years) were in control group. Flow cytometry was used to identify lymphocyte subsets. The functional state of the humoral immunity was assessed by measuring concentration of immunoglobulins IgA, IgG, IgM by using radial immunodiffusion in the gel. According to our data obtained, patients in group 1 had changes found only in the local blood flow such as prominent lymphocytopenia (decreased level of CD3+ and CD4+ lymphocytes). Patients in group 2 were featured with more pronounced changes: increased lymphocyte level, CD3+, CD4+/CD8+, IgA and lower levels of IgM, IgG in peripheral blood; high average leukocyte and lymphocyte count, IgA and reduced levels of CD3+, CD4+, CD4+/CD8+, IgM and IgG in the local area. It can be concluded that altered parameters in the local blood flow are of primary origin in mild PVV being characterized by disturbed cellular immune arm. Along with increasing disease severity, the host compensatory capabilities decline, which is manifested by pronounced combined immunodeficiency at the level of local blood flow less evident at peripheral level. These results may contribute to a more accurate assessment of intensity and dynamics of PVV progression to optimize diagnostics and immunological correction.

A mathematical model of protein subunits COVID-19 vaccines.

We consider a general mathematical model for protein subunit vaccine with a focus on the MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2, and use the model to study immunological outcomes in the humoral and cell-mediated arms of the immune response from vaccination. The mathematical model is fit to vaccine clinical trial data. We elucidate the role of Interferon-γ and Interleukin-4 in stimulating the immune response of the host. Model results, and results from a sensitivity analysis, show that a balance between the TH1 and TH2 arms of the immune response is struck, with the TH1 response being dominant. The model predicts that two-doses of the vaccine at 28 days apart will result in approximately 85% humoral immunity loss relative to peak immunity approximately 6 months post dose 1.

The crossroad between autoimmune disorder, tissue remodeling and cancer of the thyroid: The long pentraxin 3 (PTX3).

Thyroid is at the crossroads of immune dysregulation, tissue remodeling and oncogenesis. Autoimmune disorders, nodular disease and cancer of the thyroid affect a large amount of general population, mainly women. We wondered if there could be a common factor behind three processes (immune dysregulation, tissue remodeling and oncogenesis) that frequently affect, sometimes coexisting, the thyroid gland. The long pentraxin 3 (PTX3) is an essential component of the humoral arm of the innate immune system acting as soluble pattern recognition molecule. The protein is found expressed in a variety of cell types during tissue injury and stress. In addition, PTX3 is produced by neutrophils during maturation in the bone-marrow and is stored in lactoferrin-granules. PTX3 is a regulator of the complement cascade and orchestrates tissue remodeling and repair. Preclinical data and studies in human tumors indicate that PTX3 can act both as an extrinsic oncosuppressor by modulating complement-dependent tumor-promoting inflammation, or as a tumor-promoter molecule, regulating cell invasion and proliferation and epithelial to mesenchymal transition, thus suggesting that this molecule may have different functions on carcinogenesis. The involvement of PTX3 in the regulation of immune responses, tissue remodeling and oncosuppressive processes led us to explore its potential role in the development of thyroid disorders. In this review, we aimed to highlight what is known, at the state of the art, regarding the connection between the long pentraxin 3 and the main thyroid diseases i.e., nodular thyroid disease, thyroid cancer and autoimmune thyroid disorders.

Clonal relationships of memory B cell subsets in autoimmune mice.

Immunological memory protects our body from re-infection and it is composed of a cellular and a humoral arm. The B-cell branch with its memory B cells (MBCs), plasma cells and antibodies, formed either in a germinal centre (GC) -dependent or -independent manner, ensure that we can rapidly mount a recall immune response. Previous work in immunised wildtype (WT) mice have identified several subsets of MBCs whereas less is known under autoimmune conditions. Here, we have investigated the heterogeneity of the MBC compartment in autoimmune mouse models and examined the clonal relationships between MBC subsets and GC B cells in one of the models. We demonstrate the presence of at least four different MBC subsets based on their differential expression pattern of CD73, CD80 and PD-L2 in surrogate light chain-deficient (SLC-/-), MRL+/+ and MRLlpr/lpr mice, where most of the MBCs express IgM. Likewise, four MBC subsets could be identified in WT immunised mice. In SLC-/- mice, high-throughput sequencing of Ig heavy chains demonstrates that the two CD73-positive subsets are generally more mutated. Lineage tree analyses on expanded clones show overlaps between all MBC subsets and GC B cells primarily in the IgM sequences. Moreover, each of the three IgM MBC subsets could be found both as ancestor and progeny to GC B cells. This was also observed in the IgG sequences except for the CD73-negative subset. Thus, our findings demonstrate that several MBC subsets are present in autoimmune and WT mice. In SLC-/- mice, these MBC subsets are clonally related to each other and to GC B cells. Our results also indicate that different MBC subsets can seed the GC reaction.

Immunogenicity evaluation of a novel virus-like particle vaccine candidate against SARS-CoV-2 in BALB/c.

The coronavirus disease (COVID-19) pandemic has imposed deployment of an effective vaccine as a worldwide health priority. The new variants of SARS-CoV-2 have also brought serious concerns due to virus eradiation hesitancy. In this study, we evaluated the protective immune system activity of a recombinant viral vector-based vaccine candidate encoding a fusion spike, membrane and nucleocapsid proteins, Spike (528-1273aa)-M-N, in BALB/c via two different routes of delivery, intranasal and subcutaneous. The immune responses were then assessed through specific SARS-CoV-2 antibodies, interleukin and granzyme B secretion. The outcomes showed that the IgG titer and IgA secretion was higher in intranasal route in comparison with the subcutaneous, and what is more, a higher titer of IL-4 was detected through the intranasal route, whereas IFN-γ was highly induced via the subcutaneous route. The cytotoxic cell activities were mostly achieved via subcutaneous route immunization. Vaccination with the target antigen is immunogenic and led to induction of specific antibodies. Both humoral and cellular immunity arms were well activated in immunized mice, especially through intranasal route with detectable IgA and IgG. Therefore, implication of the platform as a potential vaccine candidate has potential as a future prophylactic vaccine that guarantees further investigations for the assessment of its immunogenicity in humans.

Impact of xenogenic mesenchimal stem cells secretome on a humoral component of the immune system

Objective. To determine such parameters of humoral immunity as the number of antibody-forming cells and the titer of antibodies in the blood under the action of the whole fraction of the secretome of xenogenic mesenchymal stem cells in mice with a normal immune status and mice with secondary immunodeficiency caused by the hydrocortisone acetate. Methods. The following methods were used in the study: isolation of mesenchymal stem cells from the bone marrow of cattle; culturing of isolated cells and obtaining a conditioned medium containing exometabolites with subsequent purification; modeling of secondary immunodeficiency in mice; setting up a test on mice to determine the number of antibody-forming cells in the spleen and hemagglutinin titers in blood serum; statistical data analysis. Results. A study of the impact of the secretome of xenogenic mesenchymal stem cells (MSCs) on the humoral arm of immunity in mice with a normal immune status showed a significant increase in the antibody-forming cells count compared with the control by 3.4-8.8 times when administered intramuscularly and by 4.2-5.4 times when administered subcutaneously (at the same time, in the group of the reference drug, the increase was four times), as well as an increase in the titer of hemagglutinins concerning the control by 25-95% when administered intramuscularly and 32-52% when administered subcutaneously (the reference drug Thymalin was at the control level). With intramuscular administration, a clear relationship was observed between the concentration of MSC secretome and the biological effect (high significant positive correlation: ρ = 0.99, p ≤ 0.05 for calculating AFC; ρ = 0.97, p ≤ 0.05 for HA titer). Lethality in the group of animals what administered only hydrocortisone acetate (HCA, positive control) was 100%. The use of the mesenchymal stem cells secretome increased the survival of animals by 50% by stimulating the formation of the required number of antibody-forming cells and antibody titer, except for the subcutaneous route of administration (at the level of immunized control, animals with a normal immune status). The reference drug showed a result at a level significantly lower than the immunized control. The antibody titer with the subcutaneous route of administration of secretion of MSCs was significantly lower than the immunized control but significantly higher concerning the reference drug. Conclusion. The administration of the secretome of xenogenic mesenchymal stem cells stimulated the humoral arm of immunity as same in mice with normal immune status as in mice with secondary immunodeficiency. The data obtained supplement the information on the introduction of live mesenchymal stem cells. Live allogeneic MSCs have a suppressive effect on B cells, while xenogenic MSCs cause a response on themselves. The secretome of xenogenic MSCs does not contain surface immunogenic molecules that are carried by living cells, but nevertheless increases the activity of the humoral component of immunity. The mechanisms of this effect require further study.

Intracellular trafficking of antigen in B lymphocytes

B cells are an essential part of the adaptive immune system by providing the humoral arm of the response, and specific and long-lasting protection against a pathogens. Successful antigen internalization, intracellular trafficking, processing and presentation are critical for full B cell activation and high-affinity antibody production. Despite the fundamental importance of this pathway for the immune response, the mechanisms underlying antigen trafficking for successful processing and MHCII presentation remain unclear. By using confocal and super-resolution microscopy, we followed the traffic of internalized antigen along the endosomal pathway in B cells. To identify different vesicle populations, we took advantage of different Rab GTPases as well-known markers for early-, late-and recycling-endosomes. After internalization, antigen traffics from early-to late-endosomes and typically reaches a dense endosomal compartment located in the perinuclear region. This compartment is rich in lysosomal markers and Cathepsin-S, as well as MHCII, suggesting antigen loading compartment. Interestingly however, when we investigated the early timepoints after activation in detail, we found the antigen in peripheral endosomes with atypical features combining both early (EEA1+, Rab5+) and late (LAMP1+) markers. These endosomes also showed colocalization with Rab11, a marker of recycling-endosomes, and MHCII, suggesting that already these early peripheral vesicles could degrade antigen and load peptides on MHCII for presentation. In support of their role in antigen processing, these vesicles also contained Cathepsin-S and showed hydrolysis of antigen labeled with DQ, a dye that only fluoresces upon hydrolysis. We postulate that these early vesicles could play a role in fast antigen processing, or could promote presentation of different peptide repertoire as compared to the central, perhaps more mature, perinuclear compartment. Further studies are needed to unravel how these pathways are regulated and how their balance affects T cell help and thereby B cell fate and production of high-affinity antibodies.