TNFR2 expression is a hallmark of human memory B cells with suppressive function.

Tumor Necrosis Factor Receptor 2 (TNFR2) expression is increasingly being linked to tolerogenic immune reactions and cells with suppressor function including a subset of T-regulatory cells. B-regulatory cells play an important role in control of T-cell responses and inflammation. Recently, we described TNFR2 as a marker for IL-10-producing B cells, a hallmark of this cell subset. Here, we demonstrate that proliferation of T cells is reduced in the presence of TNFR2 positive human memory B cells generated with TLR9 ligand, while TNFR2- and TNFR2+CD27- B cells display costimulatory activity. Our data further reveal that IL-10 secretion is characteristic of IgM+ naïve and memory B cells but suppressive activity is not restricted to IL-10: (i) the inhibitory effect of TNFR2+ switched memory B cells was comparable to that exerted by TNFR2+ IgM+ memory B cells although IL-10 secretion levels in the cocultures were lower; (ii) supernatants from TNFR2+ memory B cells failed to suppress T-cell proliferation. Based on our findings, we propose that formation of Breg is a specific characteristic of human memory B cells undergoing terminal differentiation. Our data further corroborate that TNFR2 represents a viable marker for identification of memory B cells with regulatory function.

Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus.

Intracellular persistence of Staphylococcus aureus favors bacterial spread and chronic infections. Here, we provide evidence for the existence of human CD4+ and CD8+ T cell memory against staphylococcal antigens. Notably, the latter could provide a missing link in our understanding of immune control of intracellular S. aureus. The analyses showed that pulsing of monocyte-derived dendritic cells (MoDC) with native staphylococcal protein antigens induced release of Th2-associated cytokines and mediators linked to T regulatory cell development (G-CSF, IL-2 and IL-10) from both CD4+ and CD8+ T cells, thus revealing a state of tolerance predominantly arising from preformed memory T cells. Furthermore, G-CSF was identified as a suppressor of CD8+ T cell-derived IFNγ secretion, thus confirming a tolerogenic role of this cytokine in the regulation of T cell responses to S. aureus. Nevertheless, delivery of in vitro transcribed mRNA-encoded staphylococcal antigens triggered Th1-biased responses, e.g. IFNγ and TNF release from both naïve and memory T cells. Collectively, our data highlight the potential of mRNA-adjuvanted antigen presentation to enable inflammatory responses, thus overriding the existing Th2/Treg-biased memory T cell response to native S. aureus antigens.

Regulatory B cells in CVID patients fail to suppress multifunctional IFN-γ+ TNF-α+ CD4+ T cells differentiation.

Common variable immunodeficiency (CVID) refers to primary hypogammaglobulinemia with unknown pathogenesis. Although there is evidence for intrinsic B cell defects in some CVID patient groups, various abnormalities in cytokine production by T cells in CVID patients are frequently observed. Here, we demonstrate a relationship in the production of pro-inflammatory Th1 cytokines and regulatory B cells producing IL-10 between CVID patients and healthy controls. We describe CD19(+)CD24(hi)CD38(hi)IL-10(+) regulatory B cells generated after T cell stimulation of human peripheral blood lymphocytes ex vivo are able to suppress IFN-γ(+)TNF-α(+) producing CD4(+) T cells. This process is impaired in CVID patients, who present with both low numbers of CD19(+)CD24(hi)CD38(hi)IL-10(+) B cells and increased numbers of IFN-γ(+)TNF-α(+)CD4(+) T cells. Disruption of the regulatory B cell response to T cell stimulation explains the excessive T cell activation regarded as an immunoregulatory abnormality that is a frequent finding in CVID patients.

Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations.

Cryopreservation of human peripheral blood mononuclear cells (PBMC) is used in many clinical and research applications to avoid direct and on-site analysis of samples. Storage of PBMC further allows prequalification of donor cells for routine laboratory methods involving the evaluation of immune responses. Previous studies reported changes in cellular composition and phenotype of PBMC following the freezing procedure. In our 12-month follow-up study, we focused on B cells and proportional representation of B cell subpopulations during long-term storage at -80 °C. Over the 12-month period, we observed a gradual decline in B cell viability and recovery. Notably, no changes in the proportional representation of human B cell subpopulations occurred in this period and the functional response elicited by antigen and TLR9 ligand CpG remained comparable to that observed after short-term storage for one month.

A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen.

Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines.

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs.

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.

Staphylococcus aureus Protein A Induces Human Regulatory T Cells Through Interaction With Antigen-Presenting Cells.

Despite continuous exposure and development of specific immunity, Staphylococcus aureus (Sa) remains one of the leading causes of severe infections worldwide. Although innate immune defense mechanisms are well understood, the role of the T cell response has not been fully elucidated. Here, we demonstrate that Sa and one of its major virulence factors protein A (SpA) induce human regulatory T cells (Tregs), key players in immune tolerance. In human PBMC and MoDC/T cell cocultures CD4+CD25+CD127dim Tregs were induced upon stimulation with Sa and to a lower extent with SpA alone. Treg induction was strongly, but not exclusively, dependent on SpA, and independent of antigen presentation or T cell epitope recognition. Lastly, soluble factors in the supernatant of SpA-stimulated MoDC were sufficient to trigger Treg formation, while supernatants of MoDC/T cell cocultures containing Sa-triggered Tregs displayed T cell suppressive activity. In summary, our findings identify a new immunosuppressory function of SpA, which leads to release of soluble, Treg-inducing factors and might be relevant to establish colonization.

Expression of Tumor Necrosis Factor Receptor 2 Characterizes TLR9-Driven Formation of Interleukin-10-Producing B Cells.

B cell-derived interleukin-10 (IL-10) production has been described as a hallmark for regulatory function in B lymphocytes. However, there is an ongoing debate on the origin of IL-10-secreting B cells and lack of specific surface markers has turned into an important obstacle for studying human B regulatory cells. In this study, we propose that tumor necrosis factor receptor 2 (TNFR2) expression can be used for enrichment of IL-10-secreting B cells. Our data confirm that IL-10 production can be induced by TLR9 stimulation with CpG ODN and that IL-10 secretion accompanies differentiation of peripheral blood B cells into plasma blasts. We further show that CpG ODN stimulation induces TNFR2 expression, which correlates with IL-10 secretion and terminal differentiation. Indeed, flow cytometric sorting of TNFR2+ B cells revealed that TNFR2+ and TNFR2- fractions correspond to IL-10+ and IL-10- fractions, respectively. Furthermore, CpG-induced TNFR2+ B cells were predominantly found in the IgM+ CD27+ B cell subset and spontaneously released immunoglobulin. Finally, our data corroborate the functional impact of TNFR2 by demonstrating that stimulation with a TNFR2 agonist significantly augments IL-10 and IL-6 production in B cells. Altogether, our data highlight a new role for TNFR2 in IL-10-secreting human B lymphocytes along with the potential to exploit this finding for sorting and isolation of this currently ill-defined B cell subset.

Lipid moieties on lipoproteins of commensal and non-commensal staphylococci induce differential immune responses.

Lipoproteins (Lpp) of Gram-positive bacteria are major players in alerting our immune system. Here, we show that the TLR2 response induced by commensal species Staphylococcus aureus and Staphylococcus epidermidis is almost ten times lower than that induced by noncommensal Staphylococcus carnosus, and this is at least partially due to their different modifications of the Lpp lipid moieties. The N terminus of the lipid moiety is acylated with a long-chain fatty acid (C17) in S. aureus and S. epidermidis, while it is acylated with a short-chain fatty acid (C2) in S. carnosus. The long-chain N-acylated Lpp, recognized by TLR2-TLR1 receptors, silences innate and adaptive immune responses, while the short-chain N-acetylated Lpp, recognized by TLR2-TLR6 receptors, boosts it.

p53 Specifically Binds Triplex DNA In Vitro and in Cells.

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.

Monitoring of CD38high expression in peripheral blood CD8+ lymphocytes in patients after kidney transplantation as a marker of cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) infection is a life-threatening complication after solid organ transplantation. It usually appears in the first months after transplantation as a consequence of immunosuppression. The goal of this study was to evaluate the clinical significance of CD38(high)/CD3(+)8(+) percentages in the detection of CMV infection in patients after kidney transplantation. METHODS: In this retrospective study, 269 patients were monitored 2-3 months after renal transplantation for the percentage of CD38(high)/CD3(+)8(+) lymphocytes estimated by flow cytometry and for the number of CMV DNA copies in peripheral blood using a real-time polymerase chain reaction. RESULTS: CMV infection was diagnosed in 12 (4.5%) patients between the 31st and 63rd days after transplantation, and all of them had percentages of CD38(high)/CD3(+)8(+) T lymphocytes above 20%. In 4 of them, CMV DNAemia in peripheral blood was not detected, and 2 of these suffered from tissue-invasive CMV disease. In 7 patients with CMV DNAemia, the CD38(high)/CD3(+)8(+) T lymphocyte percentage did not exceed 20%, and these patients did not develop CMV infection requiring antiviral treatment. In 23 additional patients, a CD38(high)/CD3(+)CD8(+) percentage above 20% was recorded without CMV DNAemia. All of the remaining 234 patients never exceeded the arbitrary limit of 20%. The estimated sensitivity and specificity were 100% and 91% using clinical decision on the presence of CMV infection as a reference value, respectively. The estimated negative predictive value was 100%; however, the estimated positive predictive value was quite low (34%). CONCLUSIONS: The CD38(high)/CD3(+)8(+) lymphocyte percentage seems to be a useful additional diagnostic marker for CMV infection in patients after kidney transplantation, especially when patients are in the risk of a tissue-invasive disease when CMV DNA copies may not be detectable in peripheral blood.