RESUMEN

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.

Asunto(s)

Antígenos/inmunología , Proteínas de Punto de Control Inmunitario/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Regulación de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/farmacología , Proteínas de Punto de Control Inmunitario/genética , Activación de Linfocitos , Melanoma/tratamiento farmacológico , Melanoma/etiología , Melanoma/metabolismo , Melanoma/patología , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica , Linfocitos T/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Virus del Dengue/inmunología , Dengue/inmunología , Células Plasmáticas/inmunología , Animales , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/metabolismo , Reacciones Cruzadas , Dengue/patología , Dengue/virología , Modelos Animales de Enfermedad , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Humanos , Masculino , Ratones , Células Plasmáticas/metabolismo , Piel/inmunología , Piel/patología , Piel/virología , Organismos Libres de Patógenos Específicos , Virus Zika/inmunología

RESUMEN

Clearance of intracellular infections caused by Salmonella Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ-/- mice succumb rapidly to STm infections, T-bet-/- mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STm-infected IFN-γ-/- and T-bet-/- mice. In IFN-γ-/- mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet-/- mice induce significant levels of IFN-γ- after challenge. Moreover, T-bet-/- mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet-/- mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS+ granuloma formation. In wild-type mice, most bacteria are within iNOS+ granulomas, but in T-bet-/- mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS+ granulomas and prevent dissemination.

Asunto(s)

Granuloma/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Proteínas de Dominio T Box/deficiencia , Células TH1/inmunología , Animales , Granuloma/genética , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Infecciones por Salmonella/genética , Salmonella typhimurium/genética , Proteínas de Dominio T Box/inmunología

RESUMEN

Antigen capturing at the periphery is one of the earliest, crucial functions of antigen-presenting cells (APCs) to initiate immune responses. Langerhans cells (LCs), the epidermal APCs migrate to draining lymph nodes (DLNs) upon acquiring antigens. An arsenal of endocytic molecules is available to this end, including lectins and pathogen recognition receptors (PRRs). However, cutaneous LCs are poorly defined in the early neonatal period. We assessed endocytic molecules expression in situ: Mannose (CD206)-, Scavenger (SRA/CD204)-, Complement (CD2l, CDllb)-, and Fc-Receptors (CD16/32, CD23) as well as CD1d, CD14, CD205, Langerin (CD207), MHCII, and TLR4 in unperturbed epidermal LCs from both adult and early neonatal mice. As most of these markers were negative at birth (day 0), LC presence was revealed with the conspicuous, epidermal LC-restricted ADPase (and confirmed with CD45) staining detecting that they were as numerous as adult ones. Unexpectedly, most LCs at day 0 expressed CD14 and CD204 while very few were MHCII+ and TLR4+. In contrast, adult LCs lacked all these markers except Langerin, CD205, CD11b, MHCII and TLR4. Intriguingly, the CD204+ and CD14+ LCs predominant at day 0, apparently disappeared by day 4. Upon cutaneous FITC application, LCs were reduced in the skin and a CD204+MHCII+FITC+ population with high levels of CD86 subsequently appeared in DLNs, with a concomitant increased percentage of CD3+CD69+ T cells, strongly suggesting that neonatal LCs were able both to ferry the cutaneous antigen into DLNs and to activate neonatal T cells in vivo. Cell cycle analysis indicated that neonatal T cells in DLNs responded with proliferation. Our study reveals that epidermal LCs are present at birth, but their repertoire of endocytic molecules and PRRs differs to that of adult ones. We believe this to be the first description of CDl4, CD204 and TLR4 in neonatal epidermal LCs in situ. Newborns' LCs express molecules to detect antigens during early postnatal periods, are able to take up local antigens and to ferry them into DLNs conveying the information to responsive neonatal T cells.

Asunto(s)

Células de Langerhans/inmunología , Células de Langerhans/fisiología , Receptores de Superficie Celular/metabolismo , Linfocitos T/metabolismo , Animales , Animales Recién Nacidos , Movimiento Celular , Proliferación Celular , Células Epidérmicas/metabolismo , Femenino , Ganglios Linfáticos , Ratones , Ratones Endogámicos BALB C , Embarazo , Piel/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral

RESUMEN

Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-ΚB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.

Asunto(s)

Antígenos Bacterianos/metabolismo , Coinfección/inmunología , Flagelina/metabolismo , Interacciones Microbiota-Huesped/inmunología , Internalización del Virus , Células A549 , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Coinfección/microbiología , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/microbiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Pulmón/citología , Permeabilidad , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 5/agonistas , Receptor Toll-Like 5/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Virosis/inmunología , Virosis/virología

RESUMEN

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

Asunto(s)

Hígado/microbiología , Infecciones por Salmonella/complicaciones , Salmonella typhimurium/patogenicidad , Bazo/microbiología , Trombosis/microbiología , Animales , Hígado/inmunología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/microbiología , Bazo/inmunología , Bazo/patología , Trombosis/inmunología , Trombosis/patología

RESUMEN

Systemic immunization with soluble flagellin (sFliC) from Salmonella Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103+CD11b+ cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4+ T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and CD11c-cre.Irf4fl/fl mice (which have reduced numbers of cDC2, particularly intestinal CD103+CD11b+ cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103+CD11b+ cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103+CD11b+ cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103+CD11b+ cDC2 in the induction of Th2-associated responses. CD11c-cre.Irf4fl/fl mice showed a reduced primary FliC-specific Th2-associated IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103+CD11b+ cDC2.

Asunto(s)

Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Flagelina/inmunología , Animales , Antígenos CD/metabolismo , Antígeno CD11c/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunización , Inmunohistoquímica , Cadenas alfa de Integrinas/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Receptor Toll-Like 5/metabolismo

RESUMEN

Antibodies acquired after vaccination or natural infection with Gram-negative bacteria, such as invasive Salmonella enterica serovar Typhimurium, can protect against disease. Immunization with naturally shed outer membrane vesicles from Gram-negative bacteria is being studied for its potential to protect against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification, and the resulting particles, generalized modules for membrane antigens (GMMA), not only offer potential as vaccines but also can facilitate the study of B-cell responses to bacterial antigens. Here we show that the response to immunization with GMMA from S Typhimurium (STmGMMA) provides B-cell-dependent protection and induces antibodies to two immunodominant antigens, lipopolysaccharide (LPS) and porins. Antibodies to LPS O antigen (O-Ag) markedly enhance protection in the spleen, but this effect is less marked in the liver. Strikingly, IgG responses to LPS and porins develop with distinct kinetics. In the first week after immunization, there is a dramatic T-cell-independent B1b-cell-associated induction of all IgG isotypes, except IgG1, to porins but not to LPS. In contrast, production of IgG1 to either antigen was delayed and T cell dependent. Nevertheless, after 1 month, cells in the bone marrow secreting IgG against porins or LPS were present at a similar frequency. Unexpectedly, immunization with O-Ag-deficient STmGMMA did not substantially enhance the anti-porin response. Therefore, IgG switching to all antigens does not develop synchronously within the same complex and so the rate of IgG switching to a single component does not necessarily reflect its frequency within the antigenic complex.IMPORTANCE Vaccines save millions of lives, yet for some infections there are none. This includes some types of Salmonella infections, killing hundreds of thousands of people annually. We show how a new type of vaccine, called GMMA, that is made from blebs shed from the Salmonella cell wall, works to protect against infection in mice by inducing host proteins (antibodies) specifically recognizing bacterial components (antigens). The rate of development of IgG antibody to antigens within GMMA occurred with different kinetics. However, the antibody response to GMMA persists and is likely to provide prolonged protection for those who need it. These results help show how antibody responses to bacterial antigens develop and how vaccines like GMMA can work and help prevent infection.

Asunto(s)

Inmunoglobulina G/inmunología , Lipopolisacáridos/inmunología , Porinas/inmunología , Infecciones por Salmonella/prevención & control , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismo , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Femenino , Masculino , Ratones , Antígenos O/inmunología , Infecciones por Salmonella/inmunología , Vacunas contra la Salmonella/inmunología , Vacunas contra la Salmonella/uso terapéutico

RESUMEN

Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding.

Asunto(s)

Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/patología , Interferón gamma/genética , Interferón gamma/metabolismo , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Infecciones por Salmonella/complicaciones , Infecciones por Salmonella/genética , Infecciones por Salmonella/patología , Trombosis/etiología , Trombosis/genética , Trombosis/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo

RESUMEN

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Asunto(s)

Inflamación/inmunología , Grasa Intraabdominal/inmunología , Linfocitos/inmunología , Tejido Linfoide/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/inmunología , Quimiocina CXCL13/metabolismo , Citometría de Flujo , Expresión Génica/inmunología , Inflamación/genética , Inflamación/metabolismo , Grasa Intraabdominal/metabolismo , Linfocitos/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/inmunología , Células del Estroma/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T cells (T-bet(+) IFN-γ(+) CD4 T cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B-cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL-4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL-12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection-induced, Th1-mediated inflammation.

Asunto(s)

Citocinas/inmunología , Células Dendríticas/inmunología , Flagelina/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Células TH1/inmunología , Animales , Citocinas/genética , Células Dendríticas/patología , Flagelina/genética , Inmunización , Ratones , Ratones Noqueados , Infecciones por Salmonella/genética , Infecciones por Salmonella/prevención & control , Salmonella typhimurium/genética , Células TH1/patología , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunología

RESUMEN

Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKß in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.

Asunto(s)

Células Dendríticas/inmunología , Redes Reguladoras de Genes/inmunología , Homeostasis/inmunología , Tolerancia Inmunológica , FN-kappa B/inmunología , Transducción de Señal/inmunología , Animales , Autoantígenos/genética , Autoantígenos/inmunología , Autoinmunidad , Movimiento Celular , Células Dendríticas/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Quinasa I-kappa B/deficiencia , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Noqueados , Análisis por Micromatrices , FN-kappa B/genética , Piel/citología , Piel/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología

RESUMEN

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

Asunto(s)

Células Presentadoras de Antígenos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Carga Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunización , Interferón gamma/biosíntesis , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Ratones , Antígenos de Histocompatibilidad Menor , Mycobacterium tuberculosis/patogenicidad , Péptidos/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/patología

RESUMEN

BACKGROUND: The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. METHODOLOGY/PRINCIPAL FINDINGS: Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. CONCLUSION: These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

Asunto(s)

Nippostrongylus/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Infecciones por Strongylida/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antihelmínticos/sangre , Coinfección/inmunología , Citocinas/biosíntesis , Inmunización , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Linfocitos T/inmunología

RESUMEN

There are multiple, distinct B-cell populations in human beings and other animals such as mice. In the latter species, there is a well-characterized subset of B-cells known as B1 cells, which are enriched in peripheral sites such as the peritoneal cavity but are rare in the blood. B1 cells can be further subdivided into B1a and B1b subsets. There may be additional B1 subsets, though it is unclear if these are distinct populations or stages in the developmental process to become mature B1a and B1b cells. A limitation in understanding B1 subsets is the relative paucity of specific surface markers. In contrast to mice, the existence of B1 cells in human beings is controversial and more studies are needed to investigate the nature of these enigmatic cells. Examples of B1b antigens include pneumococcal polysaccharide and the Vi antigen from Salmonella Typhi, both used routinely as vaccines in human beings and experimental antigens such as haptenated-Ficoll. In addition to inducing classical T-dependent responses some proteins are B1b antigens and can induce T-independent (TI) immunity, examples include factor H binding protein from Borrelia hermsii and porins from Salmonella. Therefore, B1b antigens can be proteinaceous or non-proteinaceous, induce TI responses, memory, and immunity, they exist in a diverse range of pathogenic bacteria, and a single species can contain multiple B1b antigens. An unexpected benefit to studying B1b cells is that they appear to have a propensity to recognize protective antigens in bacteria. This suggests that studying B1b cells may be rewarding for vaccine design as immunoprophylactic and immunotherapeutic interventions become more important due to the decreasing efficacy of small molecule antimicrobials.

RESUMEN

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4(+) T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼ 30-fold increase in Sca-1(hi) progenitors and a corresponding loss of Sca-1(lo/int) subsets. Most strikingly, the capacity of donor Sca-1(hi) cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1(hi) c-kit(int) cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.

Asunto(s)

Células de la Médula Ósea/inmunología , Salmonelosis Animal/inmunología , Células Madre/inmunología , Animales , Antígenos Ly/inmunología , Células de la Médula Ósea/microbiología , Células de la Médula Ósea/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Homeostasis/inmunología , Interferón gamma/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Salmonella/inmunología , Salmonelosis Animal/patología , Células Madre/microbiología , Células Madre/patología

RESUMEN

Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid Vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.

Asunto(s)

Antígenos Bacterianos/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/microbiología , Polisacáridos Bacterianos/biosíntesis , Salmonella typhi/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/fisiología , Antígenos Bacterianos/biosíntesis , Subgrupos de Linfocitos B/trasplante , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/citología , Cavidad Peritoneal/microbiología , Peritoneo/citología , Peritoneo/inmunología , Peritoneo/metabolismo , Polisacáridos Bacterianos/inmunología , Porinas , Vacunas contra la Salmonella/administración & dosificación , Vacunas contra la Salmonella/biosíntesis , Vacunas contra la Salmonella/inmunología , Fiebre Tifoidea/inmunología , Fiebre Tifoidea/metabolismo , Fiebre Tifoidea/prevención & control

RESUMEN

Mucosal immunity is poorly activated after systemic immunization with protein Ags. Nevertheless, induction of mucosal immunity in such a manner would be an attractive and simple way to overcome the intrinsic difficulties in delivering Ag to such sites. Flagellin from Salmonella enterica serovar Typhimurium (FliC) can impact markedly on host immunity, in part via its recognition by TLR5. In this study, we show that systemic immunization with soluble FliC (sFliC) drives distinct immune responses concurrently in the mesenteric lymph nodes (MLN) and the spleen after i.p. and s.c. immunization. In the MLN, but not the spleen, sFliC drives a TLR5-dependent recruitment of CD103(+) dendritic cells (DCs), which correlates with a diminution in CD103(+) DC numbers in the lamina propria. In the MLN, CD103(+) DCs carry Ag and are the major primers of endogenous and transgenic T cell priming. A key consequence of these interactions with CD103(+) DCs in the MLN is an increase in local regulatory T cell differentiation. In parallel, systemic sFliC immunization results in a pronounced switching of FliC-specific B cells to IgA in the MLN but not elsewhere. Loss of TLR5 has more impact on MLN than splenic Ab responses, reflected in an ablation of IgA, but not IgG, serum Ab titers. Therefore, systemic sFliC immunization targets CD103(+) DCs and drives distinct mucosal T and B cell responses. This offers a potential "Trojan horse" approach to modulate mucosal immunity by systemically immunizing with sFliC.

Asunto(s)

Antígenos CD/biosíntesis , Células Dendríticas/inmunología , Flagelina/administración & dosificación , Flagelina/inmunología , Factores de Transcripción Forkhead/biosíntesis , Inmunoglobulina A/biosíntesis , Cadenas alfa de Integrinas/biosíntesis , Ganglios Linfáticos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Factores de Transcripción Forkhead/fisiología , Inmunización , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Mesenterio , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Membrana Mucosa/citología , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/patología , Infecciones por Salmonella/prevención & control , Salmonella typhimurium , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Reguladores/metabolismo

RESUMEN

Thymic atrophy is a frequent consequence of infection with bacteria, viruses, and parasites and is considered a common virulence trait between pathogens. Multiple reasons have been proposed to explain this atrophy, including premature egress of immature thymocytes, increased apoptosis, or thymic shutdown to prevent tolerance to the pathogen from developing. The severe loss in thymic cell number can reflect an equally dramatic reduction in thymic output, potentially reducing peripheral T cell numbers. In this study, we examine the relationship between systemic Salmonella infection and thymic function. During infection, naive T cell numbers in peripheral lymphoid organs increase. Nevertheless, this occurs despite a pronounced thymic atrophy caused by viable bacteria, with a peak 50-fold reduction in thymocyte numbers. Thymic atrophy is not dependent upon homeostatic feedback from peripheral T cells or on regulation of endogenous glucocorticoids, as demonstrated by infection of genetically altered mice. Once bacterial numbers fall, thymocyte numbers recover, and this is associated with increases in the proportion and proliferation of early thymic progenitors. During atrophy, thymic T cell maturation is maintained, and single-joint TCR rearrangement excision circle analysis reveals there is only a modest fall in recent CD4(+) thymic emigrants in secondary lymphoid tissues. Thus, thymic atrophy does not necessarily result in a matching dysfunctional T cell output, and thymic homeostasis can constantly adjust to systemic infection to ensure that naive T cell output is maintained.

Asunto(s)

Recuperación de la Función/inmunología , Infecciones por Salmonella/inmunología , Timo/inmunología , Timo/patología , Animales , Atrofia , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Ratones , Infecciones por Salmonella/patología , Infecciones por Salmonella/fisiopatología , Salmonella typhimurium/inmunología , Timo/microbiología

RESUMEN

Phylogeny shows that CD4 T cell memory and lymph nodes coevolved in placental mammals. In ontogeny, retinoic acid orphan receptor (ROR)γ-dependent lymphoid tissue inducer (LTi) cells program the development of mammalian lymph nodes. In this study, we show that although primary CD4 T cell expansion is normal in RORγ-deficient mice, the persistence of memory CD4 T cells is RORγ-dependent. Furthermore, using bone marrow chimeric mice we demonstrate that LTi cells are the key RORγ-expressing cell type sufficient for memory CD4 T cell survival in the absence of persistent Ag. This effect was specific for CD4 T cells, as memory CD8 T cells survived equally well in the presence or absence of LTi cells. These data demonstrate a novel role for LTi cells, archetypal members of the innate lymphoid cell family, in supporting memory CD4 T cell survival in vivo.

Asunto(s)

Memoria Inmunológica , Tejido Linfoide/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Inmunidad Adaptativa/genética , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Inmunidad Innata/genética , Memoria Inmunológica/genética , Tejido Linfoide/citología , Tejido Linfoide/trasplante , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Quimera por Radiación/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/patología