RESUMEN
Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.
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Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Células Dendríticas/citología , Escherichia coli/inmunología , Lectinas Tipo C/metabolismo , Monocitos/citología , Receptores de Superficie Celular/metabolismo , Animales , Presentación de Antígeno , Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Selectina L/inmunología , Lectinas Tipo C/inmunología , Receptores de Lipopolisacáridos/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/inmunología , Receptores CCR7/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunologíaRESUMEN
BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.
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Reacciones Cruzadas , Inmunoterapia , Receptor de Muerte Celular Programada 1 , Animales , Humanos , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Ratones , Reacciones Cruzadas/inmunología , Inmunoterapia/métodos , Concentración de Iones de Hidrógeno , Neoplasias/inmunología , Neoplasias/terapia , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Epítopos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Ratones Endogámicos C57BL , FemeninoRESUMEN
Malaria blood-stage parasite is a critical pathogenic stage responsible for serious adverse outcomes in pregnant women and their neonates. Immunoglobulin G (IgG) antibody responses specific to various asexual blood-stage antigens were well reported in non-pregnant individuals. However, little is still known during placental malaria. To assess the antibody responses specific to Plasmodium falciparum-derived MSP3 and UB05 malaria vaccine candidates in mother-neonate couples, mother's peripheral blood and neonate's cord blood samples were collected at delivery. After malaria diagnostic, plasma levels of IgG and IgG subclass responses specific to UB05, MSP3 and UB05-MSP3 were determined using ELISA. As outcomes, both mothers and neonates had significantly higher IgG responses to UB05 and UB05-MSP3 compared to anti-MSP3 IgG (p < 0.05), irrespective of malaria status. Significant negative correlations were observed between IgG levels specific to the three antigens and parasitaemia (p < 0.01). Anti-UB05 and anti-UB05-MSP3 IgG levels in neonates showed a significant positive correlation with the corresponding mothers' antibodies (rs = 0.25 with p = 0.04; rs = 0.31 with p = 0.01, respectively). UB05MSP3-specific IgG3 and IgG1 subclass responses were significantly higher than the IgG4 subclass (p < 0.01). The neonates IgG1 and IgG3 levels positively correlated with the corresponding antibody subclasses of mothers. These findings suggest an association between UB05 and UB05-MSP3-specific antibody responses and malaria control during pregnancy. Maternal-foetal transfer of MSP3 and UB05-specific IgG occurs during pregnancy, suggesting the interest in the future malaria vaccination strategies in pregnant women to generate early protective immunity in baby against malaria.
RESUMEN
Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.
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Moléculas de Adhesión Celular/metabolismo , Rechazo de Injerto/prevención & control , Trasplante de Corazón , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Receptores de Superficie Celular/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/genética , Células Cultivadas , Factores de Transcripción Forkhead/metabolismo , Rechazo de Injerto/etiología , Tolerancia Inmunológica , Interleucina-10/metabolismo , Lectinas Tipo C/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Receptores de Superficie Celular/genética , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Tolerancia al Trasplante , Regulación hacia ArribaRESUMEN
BACKGROUND: Serum histamine, immunoglobulin E, and tryptase are markers of allergic diseases. Despite the reported association between migraine and allergic diseases, differences in these marker levels between episodic and chronic migraines remain unelucidated. METHODS: We investigated serum histamine, immunoglobulin E, and tryptase levels in 97 and 96 participants with episodic migraine and chronic migraine, respectively, and 56 controls according to the presence of allergic diseases. RESULTS: Serum histamine levels in episodic migraine (median and interquartile ranges, 0.78 [0.65-1.25] ng/mL, p < 0.001) and chronic migraine (0.89 [0.67-1.28] ng/mL, p < 0.001) participants were significantly lower than those in healthy controls (1.19 [0.81-2.08] ng/mL) among the 160 participants without allergic diseases. Serum immunoglobulin E levels in episodic migraine and chronic migraine participants with allergic diseases negatively correlated with headache frequency (correlation coefficient = -0.263, p = 0.017). Serum histamine levels in participants with allergic diseases and serum immunoglobulin E levels in participants without allergic diseases were not significantly different among episodic migraine, chronic migraine, and control groups. Serum tryptase levels did not significantly differ among episodic migraine, chronic migraine, and control participants with and without allergic diseases. CONCLUSIONS: Altered serum histamine and immunoglobulin E levels in episodic migraine and chronic migraine and different profiles concerning allergic diseases suggest the involvement of allergic mechanisms in migraine pathogenesis.
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Hipersensibilidad , Trastornos Migrañosos , Humanos , Histamina , Triptasas , Inmunoglobulina ERESUMEN
BACKGROUND: Individuals with migraine present ictal elevation of endothelin-1 levels. Migraine can be subclassified into episodic migraine and chronic migraine. Apart from the inconsistent reports on interictal endothelin-1 levels, most studies did not distinguish between episodic migraine and chronic migraine. METHODS: We measured plasma endothelin-1 levels in participants with episodic migraine (n = 87), with chronic migraine (n = 88), and controls (n = 50). RESULTS: Interictal endothelin-1 levels were not significantly different among participants with episodic migraine, those with chronic migraine, and controls (pg/ml, median and interquartile range, 10.19 [7.76-13.69] vs. 9.25 [6.91-10.73] vs. 9.46 [7.00-14.19], p = 0.131). After excluding participants with fibromyalgia (n = 50) and medication-overuse headache (n = 21), endothelin-1 levels were still similar among groups (10.51 [7.96-14.10] vs. 9.24 [6.96-10.81] vs. 9.46 [7.00-14.19], p = 0.230). Endothelin-1 levels did not significantly differ among participants with migraine with aura, those with migraine without aura, and controls (9.36 [4.26-13.35] vs. 9.50 [7.23-13.02] vs. 9.46 [7.00-14.19], p = 0.975). There was no significant association of endothelin-1 with headache intensity (mild, 8.99 [8.99-8.99] vs. moderate, 8.71 [6.85-12.15] vs. severe, 9.55 [7.23-13.13], p = 0.517) or headache frequency per month (exponential and 95% confidence interval, 0.709 [0.296-1.698], p = 0.440) in participants with episodic migraine and chronic migraine. CONCLUSIONS: Interictal plasma endothelin-1 level is an unlikely marker for episodic migraine and chronic migraine.
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Cefaleas Secundarias , Trastornos Migrañosos , Biomarcadores , Endotelina-1 , Cefalea , HumanosRESUMEN
BACKGROUND: Allergic asthma was typically considered as an inflammatory disease mediated by type 2 immunity. However, recent studies revealed that asthma is a complex disease displaying a variety of phenotypes and endotypes. OBJECTIVE: We examined cellular phenotypes in the mouse model of allergic asthma sensitized with different adjuvants. The aim of our study was to determine immunologic cellular characteristics in mouse asthma models induced by ovalbumin (OVA) and a variety of adjuvants. METHODS: Mice were sensitized intraperitoneally with the admixture of OVA and various adjuvants such as Alhydrogel (alum), papain, lipopolysaccharide (LPS), or CpG, and subsequently challenged with OVA intranasally. The cells in bronchoalveolar lavage (BAL) fluid, lung, and mediastinal lymph node (mLN) were examined by flow cytometric analyses. RESULTS: In the lung and BAL fluid, the highest eosinophil levels were observed in the alum group while the highest neutrophil levels were detected in the LPS group. Meanwhile, the LPS group exhibited the most elevated levels of both RORγt+ innate lymphoid cells (ILCs) and IL-17A+ Th cells in the lung and mediastinal lymph node. In the lung, the number of T-bet+ ILCs was highest in the papain group whereas the number of IFN-γ+ Th cells was highest in the CpG group. CONCLUSIONS: Notable variances are found in the composition of immune cells and expression of cytokines at the site of pathogenesis among the different mouse models of allergic asthma created by the sensitization with different adjuvants.
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Asma , Lipopolisacáridos , Adyuvantes Inmunológicos , Animales , Asma/etiología , Líquido del Lavado Bronquioalveolar , Citocinas , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Inflamación , Pulmón/patología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ovalbúmina , Papaína/metabolismoRESUMEN
Early events in atherosclerosis occur in the aortic intima and involve monocytes that become macrophages. We looked for these cells in the steady state adult mouse aorta, and surprisingly, we found a dominance of dendritic cells (DCs) in the intima. In contrast to aortic adventitial macrophages, CD11c(+)MHC II(hi) DCs were poorly phagocytic but were immune stimulatory. DCs were of two types primarily: classical Flt3-Flt3L signaling-dependent, CD103(+)CD11b(-) DCs and macrophage-colony stimulating factor (M-CSF)-dependent, CD14(+)CD11b(+)DC-SIGN(+) monocyte-derived DCs. Both types expanded during atherosclerosis. By crossing Flt3(-/-) to Ldlr(-/-) atherosclerosis-prone mice, we developed a selective and marked deficiency of classical CD103(+) aortic DCs, and they were associated with exacerbated atherosclerosis without alterations in blood lipids. Concomitantly, the Flt3(-/-)Ldlr(-/-) mice had fewer Foxp3(+) Treg cells and increased inflammatory cytokine mRNAs in the aorta. Therefore, functional DCs are dominant in normal aortic intima and, in contrast to macrophages, CD103(+) classical DCs are associated with atherosclerosis protection.
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Aterosclerosis/inmunología , Células Dendríticas/inmunología , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Antígenos CD/metabolismo , Aorta/efectos de los fármacos , Aorta/inmunología , Aterosclerosis/genética , Aterosclerosis/patología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Regulación de la Expresión Génica/inmunología , Procedimientos de Reducción del Leucocitos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Yersinia pseudotuberculosis is a Gram-negative enteropathogen and causes gastrointestinal infections. It disseminates from gut to mesenteric lymph nodes (MLNs), spleen, and liver of infected humans and animals. Although the molecular mechanisms for dissemination and infection are unclear, many Gram-negative enteropathogens presumably invade the small intestine via Peyer's patches to initiate dissemination. In this study, we demonstrate that Y. pseudotuberculosis utilizes its lipopolysaccharide (LPS) core to interact with CD209 receptors, leading to invasion of human dendritic cells (DCs) and murine macrophages. These Y. pseudotuberculosis-CD209 interactions result in bacterial dissemination to MLNs, spleens, and livers of both wild-type and Peyer's patch-deficient mice. The blocking of the Y. pseudotuberculosis-CD209 interactions by expression of O-antigen and with oligosaccharides reduces infectivity. Based on the well-documented studies in which HIV-CD209 interaction leads to viral dissemination, we therefore propose an infection route for Y. pseudotuberculosis where this pathogen, after penetrating the intestinal mucosal membrane, hijacks the Y. pseudotuberculosis-CD209 interaction antigen-presenting cells to reach their target destinations, MLNs, spleens, and livers.
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Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/microbiología , Endocitosis , Interacciones Huésped-Patógeno , Lectinas Tipo C/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/microbiología , Receptores de Superficie Celular/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Animales , Adhesión Bacteriana , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Unión Proteica , Yersiniosis/microbiología , Yersiniosis/patología , Yersiniosis/fisiopatologíaRESUMEN
Salmonella enterica serovar Typhimurium, a Gram-negative bacterium, can cause infectious diseases ranging from gastroenteritis to systemic dissemination and infection. However, the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated. A study indicated that using the lipopolysaccharide (LPS) core as a ligand, S Typhimurium was able to bind human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (hCD209a), an HIV receptor that promotes viral dissemination by hijacking antigen-presenting cells (APCs). In this study, we showed that S Typhimurium interacted with CD209s, leading to the invasion of APCs and potentially the dissemination to regional lymph nodes, spleen, and liver in mice. Shielding of the exposed LPS core through the expression of O-antigen reduces dissemination and infection. Thus, we propose that similar to HIV, S Typhimurium may also utilize APCs via interactions with CD209s as a way to disseminate to the lymph nodes, spleen, and liver to initiate host infection.
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Moléculas de Adhesión Celular/fisiología , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/fisiología , Salmonella typhimurium/patogenicidad , Animales , Células Presentadoras de Antígenos/microbiología , Femenino , Interacciones Huésped-Patógeno , Humanos , Lipopolisacáridos/fisiología , Mananos/farmacología , Ratones , Ratones Endogámicos C57BL , Antígenos O/fisiología , Ganglios Linfáticos Agregados/fisiología , Fagocitosis , Células RAW 264.7RESUMEN
BACKGROUND: In West and Central Africa areas of endemic Loa loa infections overlap with regions of high prevalence of human immunodeficiency virus type 1 (HIV-1) infections. Because individuals in this region are exposed to filarial parasites from birth, most HIV-1 infected individuals invariably also have a history of filarial parasite infection. Since HIV-1 infection both depletes immune system and maintains it in perpetual inflammation, this can hamper Loa loa filarial parasite mediated immune modulation, leading to enhanced loaisis. METHODS: In this study we have assessed in plasma from asymptomatic anti-retroviral (ARV) naïve Loa loa microfilaraemic HIV-1 infected people the filarial antibody responses specific to a filariasis composite antigen consisting of Wbgp29-BmR1-BmM14-WbSXP. The antibody responses specific to the filariasis composite antigen was determined by enzyme linked immunosorbent assay (ELISA) in plasma from ARV naïve Loa loa microfilaraemic HIV-1 infected participants. In addition the filarial antigen specific IgG antibody subclass profiles were also determined for both HIV-1 positive and negative people. RESULTS: Both Loa loa microfilaraemic HIV-1 positive and negative individuals showed significantly higher plasma levels of IgG1 (P < 0.0001), IgG2 (P < 0.0001) and IgM (P < 0.0001) relative to amicrofilaraemic participants. A significant increase in IgE (P < 0.0001) was observed exclusively in Loa loa microfilaraemic HIV-1 infected people. In contrast there was a significant reduction in the level of IgG4 (p < 0.0001) and IgG3 (P < 0.0001) in Loa loa microfilaraemic HIV-1 infected individuals. CONCLUSIONS: Loa loa microfilaraemia in ARV naïve HIV-1 infected people through differential reduction of plasma levels of filarial antigen specific IgG3, IgG4 and a significant increase in plasma levels of filarial antigen specific IgE could diminish Loa loa mediated immune-regulation. This in effect can result to increase loaisis mediated immunopathology in antiretroviral naive HIV-1 infected people.
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Antirretrovirales/uso terapéutico , Antígenos Helmínticos/inmunología , Infecciones por VIH/tratamiento farmacológico , Loiasis/diagnóstico , Adulto , Anciano , Animales , Anticuerpos Antihelmínticos/sangre , Formación de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Infecciones por VIH/complicaciones , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Loa/inmunología , Loa/aislamiento & purificación , Loiasis/complicaciones , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Langerhans cells (LCs) are skin-resident dendritic cells (DCs) that orchestrate skin immunity. CCCTC-binding factor (CTCF) is a highly conserved DNA-binding protein that regulates higher-order chromatin organization and is involved in various gene regulation processes. OBJECTIVE: We sought to clarify a possible role for CTCF in LC homeostasis and function in vivo. METHODS: We used a conditional gene deletion mouse system to generate DC- and LC-specific CTCF-ablated mice. Short hairpin RNA-mediated RNA interference was used to silence CTCF expression in human monocyte-derived Langerhans cells. DC populations were assessed by using flow cytometry and immunofluorescence. Gene expression arrays were performed to identify genes regulated by CTCF in LCs. Contact hypersensitivity and epicutaneous sensitization responses were measured to examine the functional significance of CTCF ablation. RESULTS: DC-specific CTCF deletion led to a reduced pool of systemic DCs, with LCs most severely affected. Decreases in epidermal LC numbers were specifically associated with self-turnover defects. Interestingly, CTCF-deficient LCs demonstrated impaired migration out of the epidermis. Whole-transcriptome analyses revealed that genes that promoted cell adhesion were highly expressed, but CCR7 was downregulated in CTCF-depleted LCs. Hapten-induced contact hypersensitivity responses were more sustained in LC-specific CTCF-deficient mice, whereas epicutaneous sensitization to protein antigen was attenuated, indicating that CTCF-dependent LC homeostasis is required for optimal immune function of LCs in a context-dependent manner. CONCLUSION: Our results show that CTCF positively regulates the homeostatic pool and the efficient emigration of LCs, which are required for modulating the functional immune network of the skin.
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Dermatitis por Contacto/genética , Homeostasis/genética , Células de Langerhans/metabolismo , Proteínas Represoras/genética , Animales , Factor de Unión a CCCTC , Adhesión Celular , Movimiento Celular/genética , Movimiento Celular/inmunología , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Haptenos , Homeostasis/inmunología , Humanos , Células de Langerhans/inmunología , Células de Langerhans/patología , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Receptores CCR7/genética , Receptores CCR7/inmunología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/deficiencia , Proteínas Represoras/inmunología , Transducción de SeñalRESUMEN
Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8α(+) dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8(+) T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8(+) T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257-264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-γ and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines.
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Presentación de Antígeno/inmunología , Antígenos/inmunología , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/genética , Animales , Antígenos/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Células CHO , Cricetinae , Cricetulus , Células Dendríticas/metabolismo , Inmunidad Celular , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Improved protein-based vaccines should facilitate the goal of effective vaccines against HIV and other pathogens. With respect to T cells, the efficiency of immunization, or "immunogenicity," is improved by targeting vaccine proteins to maturing dendritic cells (DCs) within mAbs to DC receptors. Here, we compared the capacity of Langerin/CD207, DEC205/CD205, and Clec9A receptors, each expressed on the CD8(+) DC subset in mice, to bring about immunization of microbial-specific T cells from the polyclonal repertoire, using HIV gag-p24 protein as an antigen. α-Langerin mAb targeted splenic CD8(+) DCs selectively in vivo, whereas α-DEC205 and α-Clec9A mAbs targeted additional cell types. When the mAb heavy chains were engineered to express gag-p24, the α-Langerin, α-DEC205, and α-Clec9A fusion mAbs given along with a maturation stimulus induced comparable levels of gag-specific T helper 1 (Th1) and CD8(+) T cells in BALB/c × C57BL/6 F1 mice. These immune T cells were more numerous than targeting the CD8(-) DC subset with α-DCIR2-gag-p24. In an in vivo assay in which gag-primed T cells were used to report the early stages of T-cell responses, α-Langerin, α-DEC205, and α-Clec9A also mediated cross-presentation to primed CD8(+) T cells if, in parallel to antigen uptake, the DCs were stimulated with α-CD40. α-Langerin, α-DEC205, and α-Clec9A targeting greatly enhanced T-cell immunization relative to nonbinding control mAb or nontargeted HIV gag-p24 protein. Therefore, when the appropriate subset of DCs is targeted with a vaccine protein, several different receptors expressed by that subset are able to initiate combined Th1 and CD8(+) immunity.
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Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos de Superficie/inmunología , Antígenos CD8 , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Lectinas Tipo C/inmunología , Lectinas de Unión a Manosa/inmunología , Receptores de Superficie Celular/inmunología , Receptores Inmunológicos/inmunología , Células TH1/inmunología , Vacunas contra el SIDA/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Proteína p24 del Núcleo del VIH/farmacología , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Antígenos de Histocompatibilidad MenorRESUMEN
Dendritic cells are antigen-presenting cells orchestrating innate and adaptive immunity. The crucial role of transcription factors and histone modifications in the transcriptional regulation of dendritic cells has been extensively studied. However, it is not been well understood whether and how three-dimensional chromatin folding controls gene expression in dendritic cells. Here we demonstrate that activation of bone marrow-derived dendritic cells induces extensive reprogramming of chromatin looping as well as enhancer activity, both of which are implicated in the dynamic changes in gene expression. Interestingly, depletion of CTCF attenuates GM-CSF-mediated JAK2/STAT5 signaling, resulting in defective NF-κB activation. Moreover, CTCF is necessary for establishing NF-κB-dependent chromatin interactions and maximal expression of pro-inflammatory cytokines, which prime Th1 and Th17 cell differentiation. Collectively, our study provides mechanistic insights into how three-dimensional enhancer networks control gene expression during bone marrow-derived dendritic cells activation, and offers an integrative view of the complex activities of CTCF in the inflammatory response of bone marrow-derived dendritic cells.
Asunto(s)
Médula Ósea , Factor de Unión a CCCTC , Células Dendríticas , FN-kappa B , Cromatina , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.
Asunto(s)
Médula Ósea , Antígenos de Histocompatibilidad Clase II , Ratones , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Células de la Médula Ósea , Células Dendríticas , Diferenciación Celular , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.
Asunto(s)
Vacunas contra el SIDA/inmunología , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Productos del Gen gag/inmunología , VIH-1/inmunología , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Adenoviridae , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Antígenos CD/genética , Relación Dosis-Respuesta Inmunológica , Productos del Gen gag/administración & dosificación , Productos del Gen gag/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/inmunología , VIH-1/genética , Haplotipos/genética , Haplotipos/inmunología , Humanos , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/inmunología , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Inyecciones Subcutáneas , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Virus VacciniaRESUMEN
During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Células Dendríticas/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/fisiología , Factores Reguladores del Interferón/biosíntesis , Transducción de Señal/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/inmunología , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Ratones , Ratones Mutantes , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Retroviridae , Transducción de Señal/efectos de los fármacos , Transducción GenéticaRESUMEN
Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-γ- and interleukin-2-producing CD4(+) T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8(+) T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.
Asunto(s)
Antígenos CD/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Proteína p24 del Núcleo del VIH/inmunología , VIH-1/inmunología , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Monoclonales , Antígenos CD/genética , Secuencia de Bases , Reactividad Cruzada , Cartilla de ADN/genética , Proteína p24 del Núcleo del VIH/genética , VIH-1/genética , Humanos , Inmunidad Celular , Inmunidad Humoral , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Receptores de Superficie Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunologíaRESUMEN
Dendritic cells (DCs) are strategically positioned to take up antigens and initiate adaptive immunity. One DC subset expresses CD8alphaalpha in mice and is specialized to capture dying cells and process antigens for MHC class I "cross-presentation." Because CD8(+) DCs also express DEC205/CD205, which is localized to splenic T cell regions, it is thought that CD8(+) DCs also are restricted to T zones. Here, we used a new antibody to Langerin/CD207, which colabels isolated CD8(+) CD205(+) DCs, to immunolabel spleen sections. The mAb labeled discrete cells with high levels of CD11c and CD8. Surprisingly most CD207(+) profiles were in marginal zones surrounding splenic white pulp nodules, and only smaller numbers were in T cell areas, where CD205 colabeling was noted. Despite a marginal zone location, CD207(+) DCs lacked identifying molecules for 3 different types of macrophages, localized in proximity and, in contrast to macrophages, marginal zone DCs were poor scavengers of soluble and particulate substrates. After stimulation with microbial agonists, Langerin expression disappeared from the marginal zone at 6-12 h, but was greatly expanded in the T cell areas, and by 24-48 h, Langerin expression disappeared. Therefore, anti-Langerin antibodies localize a majority of CD8(+) DCs to non-T cell regions of mouse spleen, where they are distinct from adjacent macrophages.