B-cell Polyclonal Activation Research Articles

Salivary Proteomics Markers for Preclinical Sjögren’s Syndrome: A Pilot Study

Primary Sjögren’s syndrome (pSS) is a complex autoimmune disorder that particularly affects the salivary and lachrymal glands, generally causing a typical dryness of the eyes and of the mouth. The disease encompasses diverse clinical representations and is characterized by B-cell polyclonal activation and autoantibodies production, including anti-Ro/SSA. Recently, it has been suggested that autoantibody profiling may enable researchers to identify susceptible asymptomatic individuals in a pre-disease state. In this pilot study, we used mass spectrometry to analyze and compare the salivary proteomics of patients with established pSS and patients with pre-clinical SS, identifying a common protein signature in their salivary fluid. We found that several inflammatory, immunity-related, and typical acinar proteins (such as MUC5B, PIP, CST4, and lipocalin 1) were differently expressed in pSS and in pre-clinical SSA+ carriers, compared to healthy controls. This suggests that saliva may closely reflect exocrine gland inflammation from the early phases of the disease. This study confirms the value of salivary proteomics for the identification of reliable biomarkers for SS that could be identified, even in a preclinical phase of the disease.

Safety of Multiple Vaccinations and Durability of Vaccine-Induced Antibodies in an Italian Military Cohort 5 Years after Immunization

We previously examined the safety and immunogenicity of multiple vaccines administered to a military cohort, divided into two groups, the first composed of students at military schools, thus operating inside the national borders for at least 3 years, and the other formed of soldiers periodically engaged in a 9-month-long mission abroad (Lebanon). In the current study, we analyzed 112 individuals of this cohort, 50 pertaining to the first group and 62 to the second group, in order to examine the possible late appearance of side effects and to calculate the half-life of the induced antibodies. Moreover, the possible involvement of B-cell polyclonal activation as a pathogenetic mechanism for long term antibody persistence has even been explored. No late side effects, as far as autoimmunity and/or lymphoproliferation appearance, have been noticed. The long duration of the vaccine induced anti-HAV antibodies has been confirmed, whereas the antibodies induced by tetravalent meningococcal polysaccharide vaccine have been found to persist above the threshold for putative protection for a longer time, and anti-tetanus, diphtheria, and polio 1 and 3 for a shorter time than previously estimated. No signs of polyclonal B-cell activation have been found, as a possible mechanism to understand the long antibody persistence.

Close relationship between immunoglobulin secreting‐cells and Epstein–Barr virus reservoir in patients infected with HIV

Individuals infected with HIV have higher circulating Epstein-Barr virus (EBV) DNA load compared to healthy carriers. This study investigated whether level of spontaneous immunoglobulin secreting cells, one of the major hallmarks of HIV infection, is associated with an increase of EBV DNA load in PBMCs and the spontaneous EBV lytic cycle ex vivo in patients infected with HIV. Spontaneous virus production by cells infected with EBV and EBV DNA loads in PBMCs from which CD8(+) T-cells were removed were measured in 20 HIV-aviremic and 14 HIV-viremic patients. The number of circulating immunoglobulin-secreting cells (Ig-SCs) and CD8(+) T-lymphocyte activation were also investigated. Patients with detectable HIV RNA in plasma exhibited higher spontaneous ex vivo EBV secretion and higher levels of EBV DNA in PBMCs than their aviremic counterparts. In the two groups observed, a positive correlation was found between PBMCs EBV DNA viral load and Ig-SCs, CD38(bright) expression on CD8(+) T-cells and EBV DNA load in cell culture supernatants. These findings suggest that B-cell polyclonal activation and B-cell terminal differentiation into Ig-SCs may fuel EBV DNA reservoir and promote EBV production ex vivo in patients infected with HIV.

IgG4-Related Systemic Disease

IgG4-related systemic disease is now recognized as a systemic disease that may affect various organs. The diagnosis is usually made in patients who present with elevated IgG4 in serum and tissue infiltration of diseased organs by numerous IgG4+ plasma cells, in the absence of validated diagnosis criteria. We report the clinical, laboratory, and histologic characteristics of 25 patients from a French nationwide cohort. We also report the treatment outcome and show that despite the efficacy of corticosteroids, a second-line treatment is frequently necessary. The clinical findings in our patients are not different from the results of previous reports from Eastern countries. Our laboratory and histologic findings, however, suggest, at least in some patients, a more broad polyclonal B cell activation than the skewed IgG4 switch previously reported. These observations strongly suggest the implication of a T-cell dependent B-cell polyclonal activation in IgG4-related systemic disease, probably at least in part under the control of T helper follicular cells.

B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis

Background Acute infectious mononucleosis (AIM) is generally associated with a large EBV B cell reservoir cells and an intense B-cell polyclonal activation whereas the number of quiescent EBV-infected memory B cells in chronically EBV-infected healthy controls is very low. Objectives To evaluate the extent and functionality of ex vivo B-cell polyclonal activation, quantify the EBV DNA integrated in B cells, enumerate the functional EBV DNA reservoir in B cells and circulating B cells spontaneously secreting EBV antigens in AIM. Study design Circulating B cells and B cells differentiating into plamablasts and plasma cells, early (BZLF1)- and late viral antigen (gp350)-secreting-cells (SCs) were enumerated in six AIM patients and seven healthy EBV carriers. Results In vitro B-cell polyclonal activation induced 8000–24,000 BZLF1- and 1000–3000 gp350-SCs/10 6 B cells, respectively. These data suggest that only 11.1–19.5% of cells expressing BZLF1 synthesized gp350 and so completed the EBV-lytic cycle. Furthermore, circulating spontaneous BZLF1- and gp350-SCs that reflect ongoing viral replication were rare (20–120 and 10–30/10 6 B cells, respectively), and their low numbers contrasted with the high levels of circulating plasma cells (1.1–10.2% of CD19 + B cells). Conclusion The in vivo terminal-B-cell differentiation into plasma cells could unmask EBV B-cell reservoir to specific cytotoxic T-cell response and combined with a predominant abortive functional-EBV-reservoir, strongly contribute to rapid decay of cellular EBV reservoir in AIM.

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation.

Mitogenicity of the recombinant mycobacterial 27-kilodalton lipoprotein is not connected to its antiprotective effect.

We reported previously that even though immunization with the recombinant mycobacterial 27-kDa lipoprotein (r27) induced a Th1-type response in mice, the vaccinated mice became more susceptible to challenge with Mycobacterium tuberculosis. In this study we show that r27 stimulates naive splenocytes to proliferate. Acylation of r27 was crucial for this effect, since a nonacylated mutant of r27, termed r27DeltaSP, failed to stimulate splenocytes either in vitro or in vivo. Depletion experiments indicated that only B cells were proliferating in a T-cell-independent manner. We also found that r27 is recognized by TLR2, which is involved in mitogenic stimulation. Interestingly, r27 but not r27DeltaSP induced high gamma interferon levels in splenocyte supernatants, whereas no significant interleukin-2 levels were detected. Since B-cell polyclonal activation might aggravate pathogen infection, we asked whether the antiprotective effect of the r27 lipoprotein is associated with its mitogenicity. We showed that, as in the case of r27, immunization of mice with the nonmitogenic r27DeltaSP lipoprotein resulted in increased M. tuberculosis multiplication. We conclude that the antiprotective effect of the r27 lipoprotein must be linked to properties of the polypeptide portion of the lipoprotein rather than to its lipid moiety and its mitogenicity.

Role of CD8+ T Cells in Mercury‐Induced Autoimmunity or Immunosuppression in the Rat

In Brown-Norway (BN) rats mercuric chloride induces an autoimmune disease characterized by an increase in serum IgE concentration, and by the production of anti-glomerular basement membrane antibodies responsible for a glomerulonephritis with a heavy proteinuria. (i) This disease results from a B-cell polyclonal activation probably due to frequent anti-class II T cells. (ii) The self limitation observed in this model is associated with both a decrease in the frequency of anti-class II T cells and the emergence of CD8+ T cells able to suppress these autoreactive T cells. (iii) In Lewis (LEW) rats which do not develop autoimmunity, HgCl2 provokes the appearance of non-antigen-specific CD8+ T cells responsible for a depression of T-cell functions. The aim of this work was to test the effect of treatment with an anti-CD8 monoclonal antibody (MoAb) in both BN and LEW rats. Anti-CD8 MoAb-treated rats were effectively depleted in CD8+ T cells. However, neither the induction nor regulation phases of mercury-induced autoimmunity were modified in BN rats. Mercury-induced immunosuppression in LEW rats was abrogated; however, depletion in CD8+ T cells did not allow the disease to occur in that strain. Finally, CD8 depletion induced in normal BN rats the appearance of rare anti-class II T cells showing that these cells are normally present in that strain but negatively controlled by suppressor T cells.

The Epstein-Barr virus-induced production of IgE by human B cells

B cells, isolated from the blood of healthy individuals and patients allergic to pollen, produced IgE when exposed to the human B-cell polyclonal activator, Epstein-Barr virus (EBV) in vitro and placed in culture. Secreted IgM and IgE were measured using immunoenzymatic assays. No difference was seen between healthy donors and allergic patients in the amount of IgE (or IgM) secreted. Cells were placed in limiting dilution cultures in order to determine the frequency of cells producing IgE or IgM (total and pollen specific) on exposure to EBV. Again, no significant differences in EBV-driven, B-cell precursor frequencies (PF) were seen between normal and allergic individuals. EBV-driven B-cell PF for total IgM and IgE, and pollen-specific IgM and IgE secretion, were 1 450 , 1 6500 , 1 83,000 , and <1 per 2,500,000, respectively, for cells from healthy donors, and 1 140 , 1 4000 , 1 56,000 and ≤1 per 2,000,000, respectively, for cells from allergic patients. We propose that the increased IgE levels seen in atopic individuals result solely from regulatory defects, rather than an increase in the frequency of B cells committed to the secretion of IgE.

Detection and partial characterization of circulating immune complexes in hydatid disease.

Thirty sera from eight patients with disseminated or localized hydatid disease have been examined for the presence of circulating immune complexes (CICs) by the conglutinin-binding assay and for immunoglobulin levels. The highest levels of CICs were of the immunoglobulin A (IgA) class, with lower values of IgG-CIC and IgM-CIC; these results did not correlate, except for IgG, with the free immunoglobulin levels. Efforts to identify parasitic antigen(s) involved in the CIC formation with different methods have been unsuccessful. In the follow-up of each patient, CIC appeared to be better correlated to clinical conditions than to hemagglutination titers. We have concluded that the presence of CIC in hydatid disease is probably an expression of B-cell polyclonal activation and that these complexes are valuable in the clinical monitoring of the disease.

Mechanism of enhanced humoral response in rodents by a synthetic polymer.

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a "conventional" B-cell polyclonal activator.

Mechanisms of altered in vitro antibody response in the rat by a small synthetic polymer.

NED 137, a low molecular weight synthetic polymer, enhances the rat humoral immune response to cellular antigens in vivo. The effect of NED 137 on the in vitro antibody response to sheep erythrocytes was studied in normal and macrophage-depleted rat spleen cultures. The polymer stimulated the IgM antibody response of normal splenocytes, and its immunopotentiating activity was increased in macrophage-depleted spleen cell preparations. NED 137 enhanced both the primary and the secondary plaque-forming cell (PFC) response of rat splenocytes in a dose- and time-dependent manner. This stimulatory effect of the polymer was dependent on the presence of antigen and demonstrated antigen specificity. The PFC and mitogenic responses of both normal and macrophage-depleted rat splenocytes were dramatically inhibited in cultures prepared from animals pretreated with NED 137 before culture initiation. The antigen dependency, specificity and potentiation of a secondary response suggest that NED 137 acts through the triggering of the lymphoid system but not as a "conventional" B-cell polyclonal activator.

Immune function in aged mice: II. B-cell function

A loss of B-cell function in old mice was demonstrated by measuring the in vitro response of lymphoid cells to the B-cell polyclonal activator, LPS (lipopolysaccharide), and the in vivo response to the thymus-independent antigen, pneumococcal polysaccharide type III (SIII). The reduced mitogenic reactivity of lymphoid cells from old compared with young mice could not be explained by a shift in kinetics of the responding cells. When LPS cultures were carried out in the presence of colchicine, fewer cells from old mice were found to respond to the mitogenic signal. The total number of B cells assessed by labelling with either anti-immunoglobulin serum or antigen-antibody complexes was not decreased in old animals. Taken together, these results are consistent with a qualitative rather than a quantitative loss of B-cell function with age. They did not, however, exclude the possibility of depletion of an LPS-reactive sub-population of B cells. Since the number of LPS-reactive cells could not be determined directly, the antibody response of old mice to SIII was investigated. The decreased level of antibody production by old mice to SIII was not due to a shift in kinetics of the responding cells. Extracellular influences were excluded by showing that the reduced responsiveness of old spleen cells persisted after adoptive transfer into young irradiated recipients. Furthermore, pretreatment of cells from old mice with anti-Thy.1 serum and complement before transfer did not enhance their antibody-forming potential. The loss of B-cell activity with age could not, therefore, be explained in terms of an increase in T-cell-dependent suppressive effects. Support for an intrinsic defect in the B cell itself came from the demonstration of similar numbers of SIII-binding cells in normal spleens from old and young mice. Following immunisation, a shift toward low intensity binding cells was observed in spleens from both old and young mice. This shift was, however, less pronounced in the case of old cells, which is consistent with an age-related decline in transformation potential of antibody-forming-cell precursors. The conclusion was, therefore, reached that the reduction with age in B-cell as well as T-cell function is due to a qualitative rather than a quantitative defect in lymphocytes themselves.