Thymus-dependent Antigen Research Articles

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

The Expression of CD28 and Its Synergism on the Immune Response of Flounder (Paralichthys olivaceus) to Thymus-Dependent Antigen.

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (Paralichthys olivaceus); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro. fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif (121TFPPPF126) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.

Towards Clinical Translation of Hematopoietic Cell Gene Editing for Treating Hyper-IgM Type 1

Hyper-IgM Type 1 (HIGM1) is caused by mutations of CD40L, whose absence in CD4 T cells impairs signaling for B cell activation and Ig class-switching. Since unregulated CD40L expression leads to lymphoproliferations/lymphomas in the mouse model of the disease, gene correction must preserve the physiological regulation of the gene. Gene editing of either autologous T cells or hematopoietic stem cells (HSC) held promise for treating HIGM1. We developed a “one size fits all” editing strategy to insert a 5'-truncated corrective CD40L cDNA in the first intron of the native human gene, effectively making expression conditional to targeted insertion in the intended locus. By exploiting a protocol that preserves T stem memory cells (TSCM), we reproducibly obtained ~40% of editing efficiency in healthy donor and patients derived T cells, restoring regulated, although partial, CD40L surface expression. The reconstituted level of expression, however, was sufficient to fully restore helper function to B cells. In order to select, track and potentially deplete edited T cells, we coupled the corrective cDNA with a clinically compatible selector gene and confirmed that enriched T cells preserved their engraftment capacity in NSG mice. Unexpectedly, the presence of an IRES-linked downstream coding frame counteracted the shorter half-life of transcript from the edited locus, allowing replenishment of intracellular stores and surface translocation of physiological amounts of CD40L upon activation. We also tailored the CD40L editing strategy to human HSC, reaching up to 15-30% editing in HSC long term engrafting NSG mice, depending on the HSC source. We then modelled the therapeutic potential of both T cell and HSC gene therapy by infusing increasing proportions of WT murine cells, as surrogates of edited cells, in HIGM1 mice. Administration of functional T cells at clinically relevant doses in HIGM1 mice, preconditioned or not with different lymphodepleting regimens, achieved long term stable T cell engraftment and partial rescue of antigen specific IgG response and germinal center formation in splenic follicles after vaccination with a thymus dependent antigen. Remarkably, infusion of T cells from mice pre-exposed to the antigen, mimicking treatment of chronically infected patients, was effective even in absence of conditioning and protected the mice from a disease relevant infection induced by the opportunistic pathogen Pneumocystis murina. Transplantation of functional T cells admixed with an equal number of HIGM1 T cells resulted in lower vaccination response, indicating competition between WT and HIGM1 cells and implying that increasing the fraction of corrected cells in the graft by selection would improve immune reconstitution. Concerning HSC gene therapy, transplanting 25% WT cells along with HIGM1 ones in HIGM1 mice - mirroring the editing efficiencies achieved in human HSC - rescued antigen specific IgG response and established protection from pathogen comparably to T cell therapy. These findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T cell as competitive strategy to HSC gene therapy, because of more straightforward translation, lower safety challenges and potentially comparable clinical benefits. We thus embarked in assessing GMP compliant reagents and protocols for T cell activation, culture and editing and developed a scalable manufacturing process. Optimization of clinical grade culture conditions allowed further increasing editing efficiency, total cellular yield and maintenance of TSCM thus paving the way to the design of a clinical trial. DisclosuresNaldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

FCRL1 inhibits ERK: Implications on B cell homeostasis and humoral immunity.

Abstract FCRL1 is a tyrosine-phosphorylated receptor found on the surface of B cells and some dendritic cells. FCRL1 co-localizes with BCR signaling complexes following BCR engagement and augments BCR-induced calcium flux. By examination of phosphotyrosine-dependent interactions of FCRL1 with intracellular signaling partners, we have identified interactions between FCRL1-Tyr281 and GRAP, GRB2, SHIP-1, and SOS1, with a requirement for GRB2 to mediate binding of FCRL1 to SHIP-1 and SOS1. Examination of MAP kinase signaling downstream of FCRL1 revealed that FCRL1 suppresses ERK phosphorylation in B cells at a resting state and following BCR engagement. In vivo, naïve FCRL1 knockout mice accumulate more B cells in the spleen, including immature, follicular and marginal zone cells, implicating a potential role for FCRL1 in B cell selection and maturation. By contrast, after immunization with thymus-dependent and independent antigens, FCRL1 knockout mice show impaired production of antigen-specific antibodies indicating an essential role for FCRL1 in humoral immune responses.

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder (Paralichthys olivaceus).

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder’s CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.

Anti-bacterial antibodies in multiple myeloma patients at disease presentation, in response to therapy and in remission: implications for patient management

Multiple myeloma (MM) is associated with increased risk of infection, but little is known regarding antibody levels against specific bacteria. We assessed levels of polyclonal immunoglobulin and antibacterial antibodies in patients recruited to the TEAMM trial, a randomised trial of antibiotic prophylaxis at the start of anti-myeloma treatment. Polyclonal IgG, IgA and IgM levels were below the reference range in 71%, 83% and 90% of 838 MM patients at diagnosis. Anti-vaccine targeted tetanus toxoid antibodies were protective in 95% of 193 healthy controls but only 41% of myeloma patients. In healthy controls, protective antibodies against 6/12 pneumococcal serotypes, haemophilus and meningococcus A were present in 67%, 41% and 56% compared to just 15%, 21% and 17% of myeloma patients. By 1 year, myeloma patients IgG levels had recovered for 57% of patients whilst the proportion with protective levels of IgG against thymus-dependent protein antigen tetanus toxoid had changed little. In contrast the proportions of patients with protective levels against thymus independent polysaccharide antigens pneumococcus, haemophilus and meningococcus had fallen from 15 to 7%, 21 to 0% and 17 to 11%. Findings highlight the need for strategies to protect patients against bacterial infections during therapy and vaccination programmes during remission.

ЭКСПЕРИМЕНТАЛЬНОЕ ИЗУЧЕНИЕ ИММУНОТРОПНОЙ АКТИВНОСТИ АЛБЕНДАЗОЛА В НИЗКИХ ДОЗАХ

Evaluation of the immunotropic effect of albendazole in low therapeutic doses has been carried out based on testing the ability of CBA × C57BL/6 mice lines to induce a delayed-type hypersensitivity (DTH) and the synthesis of antibodies against the thymus-dependent antigen. It has been shown that albendazole at doses of 2.5 and 5.0 mg/kg had no effect on the immune response of the cellular and humoral type. The DTH indices were 10.1 ± 3.14 and 10.8 ± 2.45%, respectively, and slightly exceeded those in the control by 3.1 and 10.2%. When albendazole was administered to immunized animals at doses of 2.5 and 5.0 mg/kg, log1/2 of the blood hemagglutinin titer was 6.8 ± 0.46 and 7.0 ± 0.47, and effect indices of albendazole were 1.03 and 1.06. The titer of hemagglutinins in the blood of experimental animals exceeded that in the control by 3.0 and 6.1%, respectively. It has been shown that immunocompetent cells, which functionally provide the formation of delayed-type hypersensitivity and the synthesis of antibodies against the thymus-dependent antigen, are tolerant to albendazole. Albendazole at low therapeutic doses of 2.5 and 5.0 mg/kg with a single administration did not have immunotropic activity. At the tested doses, albendazole is not a regulator of cellular and humoral processes since it does not contribute to modulation of the functional activity of immunocompetent cells and their cooperative interaction, which are necessary for the development of an immune response of the cellular and humoral type.

Influence of CD4-1+, CD4-2+ and CD8+ T lymphocytes subpopulations on the immune response of B lymphocytes in flounder (Paralichthys olivaceus) immunized with thymus-dependent or thymus-independent antigen

In order to elucidate the influence of T lymphocytes subpopulations on B lymphocytes immune response, in this paper, CD4-1+, CD4-2+, CD8+ T lymphocytes and B lymphocytes responses to thymus-independent (TI) or thymus-dependent (TD) antigen plus immunosuppressant were investigated in flounder (Paralichthys olivaceus). The results showed that in LPS-immunized group, the percentages of CD4-1+, CD4-2+, CD8β+ T (PCD4-1+ T, PCD4-2+ T and PCD8β+ T) lymphocytes in peripheral blood leucocytes (PBLs) had no significant variations, the percentages of IgM+ B (PIgM+ B) lymphocytes and LPS-specific antibodies (LA) significantly increased and peaked at 3rd or 4th week post-injection; CsA had no inhibition on both T/B lymphocytes and LA; RaPa only suppressed the PIgM+ B lymphocytes and LA, and the inhibition maximum (Imax) were about 35% and 20%, respectively. In KLH-immunized group, the PCD4-1+, PCD4-2+ and PCD8β+ T lymphocytes significantly increased and peaked at 3rd or 5th day, successively the PIgM+ B lymphocytes and KLH-specific antibodies (KA) significantly increased to the peak at 5th week; the PCD4-1+, PCD4-2+ T and PIgM+ B lymphocytes and LA were inhibited significantly by both CsA and RaPa, and the Imax on them were 13%–33%, 11%–25%, 19%–34%, 22%–26%, respectively, while the PCD8β+ T lymphocytes showed no significant suppression. The results indicated that the suppression of PIgM+ B lymphocytes in KLH + CsA group was not directly derived from CsA, but due to the suppression of T lymphocytes, especially CD4+ T lymphocytes subpopulations. The results showed for the first time that, similar to higher vertebrates, T lymphocytes didn't respond to TI antigen, moreover, T lymphocyte subpopulations had a regulation on the immune response of B lymphocyte for TD antigen in flounder.

The Sjögren's syndrome-associated autoantigen Ro52/TRIM21 modulates follicular B cell homeostasis and immunoglobulin production.

Systemic rheumatic diseases are characterized by abnormal B cell activation with autoantibody production and hypergammaglobulinaemia. Ro52/SSA, also denoted tripartite motif (TRIM)21, is a major autoantigen in Sjögren's syndrome and systemic lupus erythematosus. Interestingly, TRIM21-deficient mice develop systemic autoimmunity with B cell-driven manifestations such as autoantibodies, hypergammaglobulinaemia and glomerulonephritis following tissue injury. The mechanisms by which TRIM21-deficiency leads to enhanced B cell activation and antibody production are, however, not well understood, and to further elucidate the role of TRIM21 in systemic autoimmunity, we investigated the B cell phenotype and antibody responses of Trim21-/- mice following immunization with thymus-dependent (TD) and thymus-independent (TI) antigens. We found that TRIM21-deficient mice developed significantly higher specific antibody titres than their wild-type counterparts upon B cell receptor (BCR) engagement by TD and TI type II antigens, and this was accompanied by an altered B cell phenotype. Furthermore, BCR cross-linking, but not anti-CD40 stimulation, in vitro resulted in a significantly higher proliferation of Trim21-/- cells. We also observed that splenic follicular B cells were expanded not only in immunized mice but also already in young, unmanipulated Trim21-/- mice, and transcriptomic analysis of these cells revealed an up-regulation of genes associated with B cell differentiation, indicating a role for TRIM21 in their regulation. In conclusion, in this study we describe a link between the rheumatic autoantigen Ro52/TRIM21 and increased antibody production associated with follicular B cell expansion, implicating a potential role for Ro52/TRIM21 in the pathogenesis of systemic autoimmune diseases.

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

ABBV-105, a selective and irreversible inhibitor of Bruton’s tyrosine kinase, is efficacious in multiple preclinical models of inflammation

Objectives: Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease.Methods: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker.Results: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy.Conclusion: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.

T Follicular Helper Cells in Autoimmune Disorders.

T follicular helper (Tfh) cells are a distinct subset of CD4+ T lymphocytes, specialized in B cell help and in regulation of antibody responses. They are required for the generation of germinal center reactions, where selection of high affinity antibody producing B cells and development of memory B cells occur. Owing to the fundamental role of Tfh cells in adaptive immunity, the stringent control of their production and function is critically important, both for the induction of an optimal humoral response against thymus-dependent antigens but also for the prevention of self-reactivity. Indeed, deregulation of Tfh activities can contribute to a pathogenic autoantibody production and can play an important role in the promotion of autoimmune diseases. In the present review, we briefly introduce the molecular factors involved in Tfh cell formation in the context of a normal immune response, as well as markers associated with their identification (transcription factor, surface marker expression, and cytokine production). We then consider in detail the role of Tfh cells in the pathogenesis of a broad range of autoimmune diseases, with a special focus on systemic lupus erythematosus and rheumatoid arthritis, as well as on the other autoimmune/inflammatory disorders. We summarize the observed alterations in Tfh numbers, activation state, and circulating subset distribution during autoimmune and some other inflammatory disorders. In addition, central role of interleukin-21, major cytokine produced by Tfh cells, is discussed, as well as the involvement of follicular regulatory T cells, which share characteristics with both Tfh and regulatory T cells.

Osteopontin-enriched formula feeding improves the T-cell-dependent humoral immune responses in infant rats

Previous studies have shown that osteopontin (OPN) can enhance infant resistance to infection. However, the underlying mechanisms remain to be explored. Here, we studied the effects of OPN on the development and functions of immune cells in infant rats fed with OPN-enriched formula (OF) compared with regular formula (RF). After 21 days feeding, the proportion of infant rats’ CD3+ T cells of lymph nodes in the OF group is significantly increased compared with the RF group. The proportion of CD4+ and CD8+ T cells of lymph nodes in the OF group is closer to the breast feeding (BF) group than to the RF group. Upon immunisation with the thymus-dependent antigen ovalbumin (OVA), the concentration of OVA-specific IgG in the OF group was significantly higher than that in the RF group. Altogether OPN-enriched infant formula feeding can promote the differentiation of CD3+ T cells and improve the T-cell-dependent humoral immune responses in infant rats.

Severe deficiency in vaccine and immune responses to Streptococcus peumoniae of growth hormone (GH) deficient mice with GHRH gene ablation

Event Abstract Back to Event Severe deficiency in vaccine and immune responses to Streptococcus peumoniae of growth hormone (GH) deficient mice with GHRH gene ablation Khalil Farhat1, 2, Gwennaëlle Bodart3, Anne-Simone Parent3, Roberto Salvatori4, Henri J. Martens2 and Vincent Geenen1* 1 Université de Liège, Belgium 2 GIGA Inflammation, Infection et Immunité, Université de Liège, Belgium 3 Centre Hospitalier Universitaire de Liège, Belgium 4 The Johns Hopkins Hospital, Johns Hopkins Medicine, United States The precise impact of the somatotrope axis upon the immune system is still highly debated. From neurons in the hypothalamic arcuate nucleus, growth hormone (GH) releasing hormone (GHRH) controls the entire GHRH/GH/IGF-1 somatotrope axis. We have previously shown that mice with generalized ablation of growth hormone (GH) releasing hormone (GHRH) gene (Ghrh-/-) have normal thymus and T-cell development, but present a marked spleen atrophy and relative B-cell lymphopenia. Therefore, in this study we have investigated vaccinal and anti-infectious responses of Ghrh-/- mice against Streptococcus (S) pneumoniae, a pathogen carrying T-independent antigens. Ghrh-/- mice were unable to trigger production of specific IgM after vaccination with either native pneumococcal polysaccharides (PPS, PPV23) or protein-PPS conjugate (PCV13). Human GH supplementation (Genotonorm 2µg/g for 5 weeks) of Ghrh-/- mice restored IgM response to PPV23 vaccine but not to PCV13 suggesting that GH could exert a specific impact on the spleen marginal zone that is strongly implicated in T-independent response against pneumococcal polysaccharides. After an intranasal administration of a sublethal dose of S. pneumoniae, wild type (WT) completely cleared bacteria after 24 h, as expected. In marked contrast, Ghrh-/- mice exhibited a dramatic susceptibility to S. pneumoniae infection with a time-dependent increase in lung bacterial load and a lethal bacteremia already after 24 h. Lungs of infected Ghrh-/- mice were massively infiltrated by inflammatory macrophages and neutrophils, while lung B cells were markedly decreased. The inflammatory transcripts signature was also significantly elevated in Ghrh-/- mice. By flow cytometry analysis, the percentage of spenic marginal and follicular B cells was signifantly decreased in the spleen of infected KO mice. The distribution of a special subset of splenic macrophages identified by an anti-macrophage receptor with collagenous structure (MARCO) was also also severely affected in the spleen of infected Ghrh-/- mice. To verify whether Ghrh-/- mice have a general susceptibility to pathogens, we infected WT and KO mice with a thymus-dependent antigen: the H1N1 murine influenza virus. Infected animals exhibit a restrained mobility from day 4 to day 10, associated with a rapid respiration and fever that reach the lowest level at day 10. No difference in body weight was observed between WT and KO mice after a sublethal dose of H1N1, and KO even gained more weight after 16 days. During a supralethal H1N1 infection, both strains reached death limit point at day 6 for Ghrh-/- mice or day 8 for WT mice. This animal model unambiguously demonstrates that the somatotrope GHRH/GH/IGF1 axis plays a vital and unsuspected role in vaccine and immunological defense against S. pneumoniae. In a translational perspective, the data indicate that spleen development and response to anti-pneumococcus vaccines could be cautiously monitored in children with isolated GH deficiency, from congenital and genetic origin in particular. Acknowledgements This study was supported by grants from Association of Specialization and Scientific Guidance (Lebanon), F.R.S-FNRS, Fonds Léon Fredericq, Wallonia and W-B Federation. VG is Research director at F.R.S-FNRS. We are grateful to L. Duwez, F. Olivier, G. Lambert and Pr P. Drion from the GIGA Animal Facility for animal management, as well as the Cell Imaging and Flow Cytometry GIGA Technological Platforms. We also thank Pfizer for kindly providing Genotonorm (recombinant hGH).

The microRNA processing subunit DGCR8 is required for immune response and germinal center formation

Abstract MicroRNAs (miRNA) are a family of single stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. The microprocessor complex subunit DGCR8, which is pivotal for the canonical processing of precursor transcripts into mature miRNAs, has been shown to be indispensable for the pro-B to pre-B cell transition in early B cell development (Brandl et al., 2016). To understand the role of DGCR8 and miRNA processing during the antigen-dependent activation of mature B cells, we deleted DGCR8 during the development of immature IgM-positive B cells into mature splenic B cells by crossing a transgenic CD23-Cre mouse with a mouse homozygous for floxed DGCR8 alleles. Analysis of LPS-activated DGCR8-deficient splenic B cells from these mice revealed a reduced abundance of mature miRNAs, impaired in vitro survival, and reduced class switch to IgG1. Furthermore, frequencies of follicular B cells in the spleen, as well as B2 B cells in the peritoneal cavity were significantly altered. Finally, mice with a B cell-specific DGCR8-deficiency failed to produce antigen-specific serum IgG antibodies and adequate numbers of GL7-positive germinal center B cells upon immunization with a thymus-dependent antigen. In summary DGCR8, and thus, most likely miRNAs are required to mount effective and specific T cell-mediated immune responses.

A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus.

Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ+CD3+ T cells and IL-17+CD4+ T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus.

Memory B cells form in aged mice despite impaired affinity maturation and germinal center kinetics

We examined whether age alters the emergence of high-affinity germinal center B (GCB) cells and switched memory B cells (swBmem) during a primary immune response to a thymus-dependent antigen, using a novel flow cytometric assay to distinguish relative BCR affinity. In young mice, high-affinity B cells predominate in the GCB pool and comprise a smaller proportion of the nascent swBmem pool two weeks after immunization. In aged mice, we observe significant reductions of high-affinity clones among GCB cells, but not nascent swBmem cells. The defect in GC affinity maturation was not overcome by providing excess carrier-specific T cells from young mice, as these cells still displayed compromised effector TFH differentiation in the aged animals. Our results suggest that B cells in aged animals have a reduced ability to prompt effector TFH differentiation, leading to a compromised GC response that results in reduced generation of high-affinity GCB and plasma cells; despite normal production of early swBmem cells.

Stimulatory Effect of Volatile Urinary Components from Intact Mice on the Proliferative Activity of Splenic Lymphoid Tissue in Irradiated Animals

Immunohistochemical and morphometric analyses have demonstrated long-distance stimulatory effects of the natural volatile components of the urine of intact mice on the proliferation of splenic lymphocytes in mice exposed to a single total γ-irradiation in a dose of 1 Gy. These results are in line with the data on stimulation of the humoral immune response to thymus-dependent antigen in irradiated mice exposed to urine specimens of intact animals.

Follicular helper T cells in immunity and systemic autoimmunity

Follicular helper T (T(FH)) cells are essential for B-cell maturation and immunoglobulin production after immunization with thymus-dependent antigens. Nevertheless, the development and function of T(FH) cells have been less clearly defined than classic CD4(+) effector T-cell subsets, including T-helper-1 (T(H)1), T(H)2 and T(H)17 cells. As such, our understanding of the genesis of T(FH) cells in humans and their role in the development of autoimmunity remains incomplete. However, evidence from animal models of systemic lupus erythematosus (SLE) and patients with systemic autoimmune diseases suggests that these cells are necessary for pathogenic autoantibody production, in a manner analogous to their role in promotion of B-cell maturation during normal immune responses. In this Review, I discuss the findings that have increased our knowledge of T(FH)-cell development and function in normal and aberrant immune responses. Such information might improve our understanding of autoimmune diseases, such as SLE, and highlights the potential of T(FH) cells as therapeutic targets in these diseases.

Adaptive immunity impaired by defective lymphocyte motility and activation in mice lacking the actin-bundling protein L-plastin (121.18)

Abstract Germinal center development, critical for long-term humoral immunity, requires the migration of T and B lymphocytes to defined locations within and around the B cell follicle. Lymphocyte motility is dependent upon the linkage of extracellular adhesive and chemotactic cues to intracellular changes in the actin cytoskeleton. The molecular mechanisms linking extracellular cues and the actin cytoskeleton are not yet fully defined. We have recently identified the actin-bundling protein L-plastin as a requisite intermediary in both B and T lymphocyte motility. L-plastin is required for establishment of polarity following chemokine stimulation, for the integrin-mediated enhancement of transmigraton, and for the phsophorylation of the integrin-associated kinase Pyk-2. L-plastin is also required for mature synapse formation in T cells. In the absence of L-plastin, cell-intrinsic disruptions of T and B cell motility and activation lead to reduced follicular helper T cell generation, diminished germinal center formation and diminished antibody production to both thymus-dependent and thymus-independent antigens. Probing the specific defects in T and B cell location, activation, and survival in L-plastin-deficient mice will reveal novel insights into critical cell:cell interactions during the germinal center reaction. L-plastin thus serves as a critical bridge between adhesive and chemotactic signals and the morphological changes in actin required for T and B cell function.