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Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG+ PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM+ PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67+) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNF alpha and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response. CONACYTConsejo Nacional de Ciencia y Tecnologia (CONACyT) [61529]; SIP-IPN [20182152, 20195737, 20200594]; SAGARPA-CONACYTConsejo Nacional de Ciencia y Tecnologia (CONACyT) [2017-02-291311]; CONACYT scholarshipConsejo Nacional de Ciencia y Tecnologia (CONACyT); PIFI-IPN scholarship; COFAA-IPN; SIP-EDI; SNI-CONACYTConsejo Nacional de Ciencia y Tecnologia (CONACyT) This work was funded by CONACYT 61529, SIP-IPN 20182152, 20195737, 20200594, SAGARPA-CONACYT 2017-02-291311. EA-C was supported by CONACYT and PIFI-IPN scholarships. MA-A, ER, ZG-L, and AC-R were supported by fellowships from COFAA-IPN, SIP-EDI, and SNI-CONACYT.

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro. To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a “vaccine” to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 (P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19+CD69+) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.

Dengue is a global health problem without current specific treatment nor safe vaccines available. While severe dengue is related to pre-existing non-neutralizing dengue virus (DENV) antibodies, the role of T cells in protection or pathology is unclear. Using cutaneous DENV infection in immunocompetent mice we previously showed the generation of PNA+ germinal centers (GCs), now we assessed the activation and proliferation of B and T cells in draining lymph nodes (DLNs). We found a drastic remodelling of DLN compartments from 7 to 14 days post-infection (dpi) with greatly enlarged B cell follicles, occupying almost half of the DLN area compared to ~24% in naive conditions. Enormous clusters of proliferating (Ki-67+) cells inside B follicles were found 14 dpi, representing ~33% of B cells in DLNs but only ~2% in non-infected mice. Inside GCs, we noticed an important recruitment of tingle body macrophages removing apoptotic cells. In contrast, the percentage of paracortex area and total T cells decreased by 14–16 dpi, compared to controls. Scattered randomly distributed Ki-67+ T cells were found, similar to non-infected mice. CD69 expression by CD4+ and CD8+ T cells was minor, while it was remarkable in B cells, representing 1764.7% of change from basal levels 3 dpi. The apparent lack of T cell responses cannot be attributed to apoptosis since no significant differences were observed compared to non-infected mice. This study shows massive B cell activation and proliferation in DLNs upon DENV infection. In contrast, we found very poor, almost absent CD4+ and CD8+ T cell responses.

Tuberculosis is one of the leading causes of human morbidity and mortality. Mycobacterium tuberculosis (Mtb) employs different strategies to evade and counterattack immune responses persisting for years. Mast cells are crucial during innate immune responses and help clear infections via inflammation or by direct antibacterial activity through extracellular traps (MCETs). Whether Mtb induce MCETs production is unknown. In this study, we report that viable Mtb did not induce DNA release by mast cells, but heat-killed Mtb (HK-Mtb) did. DNA released by mast cells after stimulation with HK-Mtb was complexed with histone and tryptase. MCETs induced with PMA and HK-Mtb were unable to kill live Mtb bacilli. Mast cells stimulated with HK-Mtb induced hydrogen peroxide production, whereas cells stimulated with viable Mtb did not. Moreover, MCETs induction by HK-Mtb was dependent of NADPH oxidase activity, because its blockade resulted in a diminished DNA release by mast cells. Interestingly, catalase-deficient Mtb induced a significant production of hydrogen peroxide and DNA release by mast cells, indicating that catalase produced by Mtb prevents MCETs release by degrading hydrogen peroxide. Our findings show a new strategy employed by Mtb to overcome the immune response through inhibiting MCETs formation, which could be relevant during early stages of infection.

Allergic asthma is a chronic inflammatory disease characterized by the accumulation of eosinophils, Th2 cells and mononuclear cells in the airways, leading to changes in lung architecture and subsequently reduced respiratory function. We have previously demonstrated that CDIP-2, a chemokine derived peptide, reduced in vitro chemotaxis and decreased cellular infiltration in a murine model of allergic airway inflammation. However, the mechanisms involved in this process have not been identified yet. Now, we found that CDIP-2 reduces chemokine-mediated functions via interactions with CCR1, CCR2 and CCR3. Moreover, using bone marrow-derived eosinophils, we demonstrated that CDIP-2 modifies the calcium fluxes induced by CCL11 and down-modulated CCR3 expression. Finally, CDIP-2 treatment in a murine model of OVA-induced allergic airway inflammation reduced leukocyte recruitment and decreases production of cytokines. These data suggest that chemokine-derived peptides represent new therapeutic tools to generate more effective antiinflammatory drugs.

Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases (such as anti-phospholipid syndrome, systemic lupus erythematosus). However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with systemic lupus erythematosus and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1-, CD19-, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD-, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. In contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.

Dengue virus (DENV) is one of the most significant human viral pathogens transmitted by mosquitoes and can cause from an asymptomatic disease to mild undifferentiated fever, classical dengue, and severe dengue. Neutralizing memory antibody (Ab) responses are one of the most important mechanisms that counteract reinfections and are therefore the main aim of vaccination. However, it has also been proposed that in dengue, some of these class-switched (IgG) memory Abs might worsen the disease. Although these memory Abs derive from B cells by T-cell-dependent processes, we know rather little about the (acute, chronic, or memory) B cell responses and the complex cellular mechanisms generating these Abs during DENV infections. This review aims to provide an updated and comprehensive perspective of the B cell responses during DENV infection, starting since the very early events such as the cutaneous DENV entrance and the arrival into draining lymph nodes, to the putative B cell activation, proliferation, and germinal centers (GCs) formation (the source of affinity-matured class-switched memory Abs), till the outcome of GC reactions such as the generation of plasmablasts, Ab-secreting plasma cells, and memory B cells. We discuss topics very poorly explored such as the possibility of B cell infection by DENV or even activation-induced B cell death. The current information about the nature of the Ab responses to DENV is also illustrated.

Evasion or subversion of host immune responses have been shown for a variety of microorganisms, and this might be the case for Trichophyton rubrum, the most common pathogenic fungus causing chronic dermatophytosis in humans. Keratinocytes, the main epidermal cells, have important roles as a first defense against microbial challenges in local immune reactions. Epidermal keratinocytes express several Toll-like receptors and produce host defense peptides, cytokines and chemokines in response to various stimuli. We analyzed the expression of Toll-Like receptor TLR2, TLR4, TLR6, and Human Beta Defensin (HBD)-1, HBD-2, Interleukin IL-1b and IL-8 production, when exposing primary keratinocyte cultures to T. rubrum. We observed changes in size and granularity of keratinocytes stimulated with either whole conidia or conidial homogenates compared to other treatments. Intact conidia decreased keratinocytes’ TLR2 and TLR6 expression without affecting that of TLR4, while conidial homogenates increased the expression of these three receptors. Interestingly, whole conidia decreased HBD-1 and HBD-2 production, whereas conidial homogenate increased it. No changes were observed in IL-1b and IL-8 production after stimulation with conidia or conidial homogenate. CONCLUSIONS. Our results suggest that: 1) Keratinocytes can recognize and respond to cell wall components of T. rubrum; 2) Viable intact conidia inhibit TLR-2 and TLR6 expression and decrease HBD-1 and HBD-2 production; 3) Conidial homogenate from T. rubrum increases the expression of TLR2, TLR4 and TLR6 and induces HBD-1 and HBD-2 production; 4) Therefore, innate immune functions of keratinocytes as the first level of local skin immunity are apparently manipulated by T. rubrum, likely to ensure its establishment, persistence and survival.

This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyer's patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIM wt ). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF-α), and beta interferon (IFN-β), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response.

Although the high-risk human papillomavirus (HPV) is necessary to cause cervical cancer (CC) and its infectious origin is well recognized, neither the systemic nor the local immune responses to this virus has been well studied or understood. Because the many facets of HPV are excellently described here by others, we will focus on the immune responses of the female genital tract, especially in situ, or in natura, as Casanova argues when studying diseases (1). A general overview is provided where different elements of the innate and adaptive immunity are discussed. We highlight the very successful strategy used by HPV with a protracted and seemingly silent infection, making this virus extremely well adapted to infect humans. The counterpart of this peculiar immunological situation is that whatever immune responses (innate or adaptive, local or systemic) are induced in those chronically infected women, these responses are inefficient to cope with and especially to eradicate the virus, thus resulting in viral persistence. A further follow-up of this line of thought is that among other well-known co-factors some still uncovered complex immune alterations may underlie the enigmatic susceptibility of the minority of women permissive to the chronic HPV infection, i.e., of those who slowly progress from infection to CIN 1e2e3 and then to invasive cervical carcinoma.

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.

Dengue virus (DENV) has four serotypes, which can cause from asymptomatic disease to severe dengue. Heterologous secondary infections have been associated to a greater risk of potentially fatal dengue due to non-neutralizing memory antibodies (Abs), which facilitate the infection, such as anti-precursor membrane (prM) Abs, among other mechanisms. Usually, class-switched memory Abs are generated mainly through germinal centers (GCs). However, the cellular events underlying these Ab responses to DENV, especially during repeated/secondary infections, have been poorly studied. We wanted to know whether there is involvement of GC reactions during cutaneous DENV infection and whether there is any sort of preferential Ab responses to defined viral proteins. Intradermal DENV inoculation at a relatively low dose efficiently infects immune-competent BALB/c mice, inducing higher quantities of DENV-specific GC B cells and larger GCs than the control conditions. Interestingly, GCs exhibited as much prM as envelope (E) and non-structural 3 viral proteins in situ. Intriguingly, despite the much larger abundance of E protein than of prM protein in the virions, infected animals showed similar amounts of circulating Abs and Ag-specific GC B cells both for prM and for E proteins, even significantly higher for prM. To the best of our knowledge, there are no reports of the GC responses during DENV infection. This relatively stronger anti-prM response could be triggered by DENV to preferentially promote Abs against certain viral proteins, which might favor infections by facilitating DENV invasion of host cells. It is thus conceivably that DENV might have evolved to induce this kind of Ab responses.

Dendritic cells (DC) are often arranged in planar layers in tissues with high antigenic exposure, such as skin and mucosae. Providing an en face view, this arrangement optimizes in situ analysis regarding morphology (even of individual dendrites), topographic distribution (regular/clustered) and quantification. The few reports on human genital DC usually utilize single markers and conventional sections, restricting immunolabelling only to cell parts sectioned by the cut. To better assess DC in situ, we labelled epithelial sheets, prepared from fresh cervix biopsies, with antibodies to major histocompatibility complex (MHC)-CII, CD1a and Langerin, revealing (with each of these markers) a dense DC network in a planar-like, regular distribution. Using the hybrid capture system to detect the high-risk mucotropic human papilloma virus (HPV) group, 16 positive and five negative women were studied and the results were compared between these groups. DC frequency per area was substantially reduced (to approximately 50% for the three markers) in samples from all HPV-infected patients compared with samples from controls. Unlike HPV(-) samples, Langerin(+) DC in HPV(+) cervix exhibited a highly accentuated dendritic appearance. We believe this to be the first study using these three DC-restricted markers (Langerin, CD1a and MHC-CII) in cervical epithelial sheets from high-risk HPV(+) donors and also the first study to demonstrate the morphological and quantitative changes triggered by high-risk HPV infection. Cervical DC reduction in early, premalignant high-risk HPV infection might represent viral subversion strategies interfering with efficient antigen handling by the immune system's peripheral sentinels, the DC, perhaps hampering appropriate recruitment and subsequent development of effector (cytotoxic) T cells.

CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39-kDa glycoprotein (CD154) expressed on the surface of activated T lymphocytes and is essential for thymus-dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40-CD40 ligand interaction in vitro and in vivo by using CD154-transfected L929 cells. In vitro assays showed that CD154-L929 cells can induce on B cells: IL-4-dependent proliferation, up-regulation of CD23, CD54 and class II molecules and can also rescue WEHI-231 B cell lymphoma from anti-IgM-induced apoptosis. Interestingly, in vivo assays revealed that when CD154-L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti-erythrocyte autoantibodies. Through B lymphocyte activation with CD154-transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40-CD154 in the induction of primary responses in vivo.

Whereas CD40–CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand −/− mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand −/− mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand −/− mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-α production and was corrected by injecting recombinant TNF-α or an agonistic anti-CD40 monoclonal antibody. Thus, CD40–CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-α production and play a vital role in the initiation of acquired T cell–mediated immunity.

Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40+ lineage-negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high-level expression of major histocompatibility complex class II, the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL-2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co-localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.

Germinal center (GC) B cells readily undergo apoptosis, a tendency which can be suppressed in vitro by immobilized anti-Ig; mAb to CD40 and soluble CD23 (in synergy with IL-1 alpha) also effect rescue of GC cells from programmed cell death. In the present study, the signals which stimulate rescued GC populations to DNA synthesis have been examined and compared to those established for the activation of follicular mantle (FM) B cells. On co-culture with anti-Ig, optimal responses in FM B cells can be achieved with a combination of IL-4 and CD40 antibody; these activities also provided a modest stimulus to GC cells but, for this population, anti-Ig was ineffective at augmenting the response further. Stimulations of GC B cells were enhanced, however, when performed on a support of primary fetal lung fibroblasts; a major influence of stroma was to promote, by direct cell-cell contact, the CD40-dependent survival of GC B cells. FM B cells were relatively independent of such stromal support. In marked contrast to FM cells, GC B cells were found to respond by enhanced DNA synthesis to IL-2 even when quite low concentrations of the factor were present (IC50 = 2 U/ml). Stimulation of GC cells via this pathway was augmented almost 2-fold on the inclusion of anti-Ig whereas neither fibroblasts, IL-4, nor CD40 antibody made any additional contribution to the IL-2-dependent response. The requirements found for stimulating GC cells in vitro are discussed with reference to the signals that this population may encounter in appropriate microenvironments in vivo: the variety of options apparently available could reflect changing priorities at different stages of a developing GC response.

A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in peripheral blood cell suspensions and in cryosections of horse lymph nodes. Only the serum against peptide 2 was capable of identifying cells in suspension and in situ by immunohistochemistry, some with evident dendritic morphology. Using this approach, an immunogenic epitope exposed in CD11c was identified in cells from horse lymph node in situ.

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long-lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen-associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long-lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules on dendritic cells and macrophages, and long-lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen-presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long-lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Recent findings indicate that atherosclerosis, a chronic inflammatory process, might start during childhood. Nevertheless, the expression of inflammation-related molecules of endothelial cell isolated from healthy neonates with a strong family history of myocardial infarction (SFHMI) has been rarely analyzed.Human umbilical vein endothelial cells (HUVECs) from children with SFHMI were assessed for the expression of CD40 and CD40L, in the presence of TNF-alpha and oxLDL. The intracellular content of CD80, CXCL8 and tissue factor by HUVECs stimulated with a CD40 agonist monoclonal antibody as well as monocytes/lymphocyte adhesion to TNF-alpha-stimulated HUVECs was also evaluated.The basal expression of CD40 and CD40L was higher in SFHMI-positive HUVECs in comparison to controls. TNF-alpha and oxLDL upregulated the expression of CD40 and CD40L in SFHMI versus control HUVECs (p0.001). The intracellular expression of CXCL8, tissue factor and CD80 was also higher than in controls, and the adhesion of lymphocyte- and monocyte-like cells augmented upon TNF-alpha stimulation.It is possible that the modifications observed in the SFHMI-positive HUVECs, all of them relevant to the atherosclerosis process, may lead to early inflammatory reactions, thus contributing to the premature initiation of atherosclerotic lesions in these children.

Dendritic cells (DCs) are located at body surfaces such as the skin, respiratory and genital tracts, and intestine. To further analyze intestinal DCs, we adapted an epidermal sheet separation technique and obtained two intestinal layers, facing the lumen and serosa. Unexpectedly, immunolabeling of the layer toward the serosa revealed a regular, dense, planar network of cells with prominent dendritic morphology within the external muscular layer and with increasing frequency along the length of the intestine. Direct examination of the serosal-disposed layers showed a significant fraction of the DCs to express DEC-205/CD205, CD11c, Langerin/CD207, Fcγ receptor/CD16/32, CD14, and low levels of activation markers, CD25, CD80, CD86, and CD95. By more sensitive FACS analyses, cells from this layer contained two CD11c+populations of CD45+CD205+, CD19-leukocytes, MHC II+and MHC II-. When ovalbumin conjugated to an anti-DEC-205 antibody was injected into mice, the conjugate targeted to these DCs, which upon isolation were able to stimulate ovalbumin-specific, CD4+and CD8+T cell antigen receptor-transgenic T cells.In vivo, these DCs responded to two microbial stimuli, systemic LPS and oral live bacteria, by up-regulating CD80, CD86, DEC-205, and Langerin within 12 h. This network of DCs thus represents a previously unrecognized antigen-presenting cell system in the intestine.

Antibodies recognizing non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, triggered an autoimmune-like disease in mice. This exhibited features similar to human lupus and was induced by injecting mice either with the H308 monoclonal antibody specific to NPA, with sera from mice which already had developed the autoimmune disease, or with liposomes treated with the NPA inductors chlorpromazine or procainamide; or with these NPA inductors alone. All these procedures revealed the involvement of antibodies to non-bilayer phospholipids in inducing this autoimmune-like disease. Unraveling the mechanisms of these antibodies might contribute to a better understanding of the molecular and immunological basis of autoimmune diseases like lupus and, hopefully, towards the development of better therapeutic strategies.

SUMMARY Despite tuberculosis resurgence and extensive dendritic cell (DC) research, there are no in vivo studies evaluating DC within regional lymphoid tissue during airways infection with virulent Mycobacterium tuberculosis (Mtb) H37Rv. Using DC-specific antibodies, immunocytochemistry, flow cytometry and Ziehl–Neelsen (ZN) for bacilli staining, we searched for Mtb and DC changes within mediastinal lymph nodes, after intratracheal (ITT) inoculation of virulent Mtb. ZN and immunocytochemistry in frozen and paraffin sections of mediastinal lymph nodes identified Mtb until day 14 after ITT inoculation, associated with CD11c + and Dec205 + DC. Analysing CD11c, MHC-CII, and Dec205 combinations by flow cytometry in MLN suspensions revealed that CD11c + /MHC-CII + and CD11c + /Dec205 + DC did not increase until day 14, peaked on day 21, and sharply declined by day 28. No changes were seen in control, salineinoculated animals. The costimulatory molecules evaluated in CD11c + DCs followed a similar trend; the CD80 increase was negligible, slightly surpassed by CD40. CD86 increased earlier and the three markers peaked at day 21, declining by day 28. While antigen-specific proliferation was not evident for MLN CD4 + T cells at 2 weeks postinfection, delayed-type hypersensitivity responses upon ITT inoculation revealed that, as early as day 3 and 7, both the priming and peripheral systemic immune responses were clearly established, persisting until days 14–21. While airways infection with virulent Mtb triggers an early, systemic peripheral response maintained for three weeks, this seems dissociated from regional events within mediastinal lymph nodes, such as antigen-specific T-cell reactivity and a delay in the influx and local activation of DC.

The hallmark of immunity is antigen (Ag) recognition. From early life until death we face countless Ags; many are deleterious and come from the outside world. However, lymphocytes, the mediators of specific acquired immunity, are usually not exposed where foreign antigens are encountered. Thus a system must ferry these Ags from the periphery into inner tissues where lymphocytes comfortably reside or traffic. Clearly, translocating Ags from the periphery to the lymphoid niches is a pivotal function whereby the dendritic cell (DC) system initiates immune responses. DCs, however, are not passive Ag couriers; a dynamic process not fully understood is triggered by Ag deposition. Strategically poised at the boundaries between the inner and the outside world, in a way bridging innate and acquired immunity, DCs sample, trap, engulf, and digest Ags of very diverse origin, in what is called Ag processing. Antigenic fragments are then regurgitated, exposed at the cell surface through one of three molecular pathways [Major histocompatibility complex (MHC) CI and CII, and CD1)] responsible for an efficacious Ag presentation. Concomitantly, DC bear an arsenal of powerful costimulatory molecules [CD40; CD80; CD86; DC-specific/intercellular adhesion molecule type 3-grabbing, non-integrin (DC-SIGN)] and the potential to produce critical cytokines [chemokines, interleukin-12 (IL-12), etc.], thus ensuring the initiation and the fate of acquired immunity. The DC system is particularly efficient, not just translocating, but also transforming antigens sampled at the earlier local milieu into an immunologically accesible language, becoming an excellent reporter of the previous antigenic past, translating the conundrum posed by viruses, bacteria, proteins, etc., into T-cell receptor (TCR) ligands; a short-peptide vocabulary that T cells especially now understand. A highly dynamic process is thus triggered by encountering Ags; while these are carried and processed to be appropriately presented, DCs in turn experience a variety of changes: migratory, phenotypical, functional; all encompassed in the term maturation. Essentially, DC maturation means to change from an antigen-capturing to an antigen-presenting, T-cell-priming mode, a process whereby DCs convert antigens into efficacious immunogens, express the necessary cytokines and costimulatory molecules, thus appropriately initiating the specific, acquired clonal immunity.

Dendritic cells (DC) are cells of the hematopoletic system specialized in capturing antigens and initiating T cell-mediated immune responses. We show here that human DC generated in vitro by culturing CD34+ cord blood progenitor cells in granulocyte macrophage colony stimulating factor plus tumor necrosis factor-alpha express the Fas antigen (APO-1, CD95) and undergo apoptosis upon triggering of Fas by mAb. However, only a proportion of the cells die in response to Fas ligation, an observation that may be related to the virtual absence of the bcl-2 protein in about half of the cells. Ligation of DC CD40 by culture on CD40L-transfected fibroblastic cells up-regulates the expression of bcl-2 and, concomitantly, renders DC virtually resistant to Fas-induced apoptosis. Parallel experiments with mature, interdigitating dendritic cells (IDC) isolated from tonsils revealed that IDC express Fas but do not enter into apoptosis following Fas ligation, a finding that may be explained by their high levels of bcl-2. Thus, upon encountering antigen-specific T cells, DC become resistant to Fas-induced apoptosis, as a consequence of CD40 ligation and possibly by mechanisms associated to the up-regulation of bcl-2 protein expression.

Following advances during the past 5 years in our understanding of the molecular structure of receptors for IgE, progress has been made in elucidating the structure and function of IgE receptors and the signalling events through these receptors. IgE is not the only ligand for some of these receptors, leading to their having unexpected and interesting biological activities.

Immunoglobulin E (IgE) is central to the induction of allergic diseases through its binding to the high-affinity receptor (Fc epsilon R1) on mast cells and basophils. Crosslinking by allergens of the bound IgE leads to the release of various inflammatory mediators. IgE production by B cells requires a physical interaction with T cells, involving a number of surface adhesion molecules, as well as the soluble factors interleukin-4 (IL-4) and IL-13 (ref. 5) produced by T cells, basophils and mast cells. Here we report that, in the presence of IL-4, mast and basophilic cell lines can provide the cell contact signals that are required for IgE synthesis. The human cell lines HMC-1 (mast) and KU812 (basophilic) both express the ligand for CD40 (CD40L) which is shown to be responsible for the IgE production. Moreover, freshly isolated purified human lung mast cells and blood basophils are also shown to express CD40L and to induce IgE production. This evidence suggests that mast cells and basophils may therefore play a key role in allergy not only by producing inflammatory mediators, but also by directly regulating IgE production independently of T cells.

Immunoglobulin E (IgE) mediates many allergic responses. CD23 is a 45-kilodalton type II transmembrane glycoprotein expressed in many cell types. It is a low-affinity IgE receptor and interacts specifically with CD21, thereby modulating IgE production by B lymphocytes in vitro. In an in vivo model of an allergen-specific IgE response, administration of a rabbit polyclonal antibody to recombinant human truncated CD23 resulted in up to 90 percent inhibition of ovalbumin-specific IgE synthesis. Both Fabs and intact IgG inhibited IgE production in vitro and in vivo. Thus, CD23 participates in the regulation of IgE synthesis in vivo and so could be important in allergic disease.

BACKGROUND: CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we used Jak3(-/-) mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3(-/-) bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3(-/-) mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3(+/+)). In addition, when we analyzed the migration of Jak3(-/-) and Jak3(+/+) mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3. CONCLUSION/SIGNIFICANCE: Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway.

Following the observation of Bonnefoy et al. (J. Exp. Med. 1988. 167: 57), that the low-affinity IgE receptor (CD23) on B lymphocytes can be coupled (with the use of chemical cross-linking reagents) to major histocompatibility complex (MHC) class II DR molecules, we now report that ligands binding within the lectin-homology region of CD23 prevent B cells from stimulating allogeneic mixed lymphocyte responses. Ligands capable of blocking mixed lymphocyte responses include the anti-CD23 antibodies MHM6 and EBVCS 4 but not EBVCS 1 and 5, IgE itself, and small peptides representing sequences within the CH3 domain of IgE. The detailed topographical relationship between CD23 and MHC class II on the B lymphocyte surface was examined using dual immuno-fluorescence labeling of cells and direct visualization of the staining by confocal laser scanning microscopy. On transformed B lymphoblasts, the two antigens were seen to co-localize in discrete patches; on normal B cells which had been cultured for 2 days with interleukin 4, CD23 and MHC class II converged at a single pole which exhibited a tendency to pseudopod formation and provided a focus for homotypic cell-cell interactions. The possibility that CD23 could serve as a co-stimulatory-adhesion molecule in antigen presentation by B lymphocytes is discussed with special reference to a potential role in the regulation of IgE synthesis.

The molecules controlling IgE production are the subject of intense study in the effort to find new ways to treat allergic diseases. One candidate, the CD23 molecule, a low affinity receptor for IgE, was recently identified to interact with another molecule, named CD21, in the regulation of IgE production.

We read with interest the recent article by Mudde and colleagues (Immunol. Today, 1990, 11, 440- 443), speculating on a special role for IgE in antigen presentation. However, the emphasis may have been placed on the wrong molecule as the 'capture of antigen' is surely a feature of all classes of immuno- globulin, not just IgE! On the other hand, CD23 - which can bind IgE with low affinity - may well contrib- ute to the presentation of antigen by B cells and, perhaps, other cells 1. CD23 is spatially linked to major histocompatibility complex (MHC) class II DR 2 and ligands binding within its lectin-homology domain suppress the antigen-presenting ca- pacity of B cells 3,4. Importantly, the switching of uncommitted B cells to the production of IgE minimally requires (1) physical contact with CD4 + cells via MHC class H mol- ecules and the T-cell receptor, and (2) the interleukin 4 (IL-4) that is subsequently produced 5. We have postulated that interrupting these essential early events by surplus IgE binding to CD23 could provide negative feedback on IgE production in an ongoing response4: where anti- gen remained in excess, positive feedback would ensue after the internalization of IgE-antigen complexes. Rather than simply providing an anchor for IgE, we view CD23 functioning directly as an ac- cessory molecule during antigen presentation by B cells. Indeed, sol- uble CD23 is co-mitogenic for CD4 ÷ T cells (Ref. 6 and J.M. Bertho etal., unpublished), while the de novo ex- pression of membrane CD23 pro- motes intercellular adhesions among previously nonadherent cells 7. The most striking expression of CD23 is associated with a subset of follicular dendritic cells (FDCs) and we have suggested a role for FDC- associated CD23 in the development of germinal centre B cells 1. There is no evidence that FDCs process anti- gen; yet, in pursuit of their thesis, Mudde and colleagues seemed pre- pared to dismiss a large body of work on the B-lymphotropic proper- ties of CD23 by citing a single reference where bacterial-derived recombinant material was appar- ently inactive in a standard 'BCGF' assay 8. Recent studies demonstrate that - in synergy with IL-1 - recombinant 25 kDa CD23 derived from insect cells assists the survival of germinal centre B cells and pro- motes their morphological differen- tiation (Y-J. Liu et al., unpublished). Such synergistic activity for recom- binant CD23 and IL-1 has also been noted for the differentiation of im- mature thymocytes and CD34 + myeloid precursors 9,1°. The role of the low-affinity IgE receptor in antigen presentation is clearly a fascinating area which de- serves further study. The accumu- lated data, however, suggest that this molecule is important not only in the regulation of IgE synthesis but also in mechanisms of lymphocyte and monocyte differentiation that are quite independent of IgE.

The serum levels of IgE and the soluble cleavage product of CD23 (sCD23) were prospectively monitored for up to 1 year after transplantation in 34 patients who underwent autologous (n = 33) or syngeneic (n = 1) bone marrow transplantation (BMT). In 25 patients (74%), a transient IgE peak (two- to 2,750-fold increase) appeared in the serum 3 to 4 weeks after BMT. In 18 patients (51%), a two- to 125- fold increase in sCD23 coincided with the IgE peak. In only three patients was a sCD23 peak observed without a concomitant increase in IgE. The sCD23 increment preceded the IgE peak in each individual case. During the period of increased sCD23 serum levels, the absolute numbers of circulating B cells and other cell types expressing surface CD23 were extremely low. The biologic significance of these findings is discussed in light of present knowledge of regulation of B-cell growth and differentiation with special reference to the role of sCD23 as a multifunctional cytokine.