Participación de Galectina-8 en la inducción de la respuesta inmune

Al presente se ha acumulado evidencia suficiente como para posicionar las galectinas (Gals) dentro de los principales mediadores de la respuesta inmune innata y adaptativa. Por lo tanto, resulta crucial entender cómo las Gals participan en el control de la homeostasis celular del sistema inmune y los mecanismos subyacentes. En la presente Tesis, describimos por primera vez un efecto activador de Gal-1 sobre células T naïve, lo cual queda demostrado por la inducción de co-estimulación y proliferación, esta última inducida en forma de quimera Gal-1-8-1. El bloqueo de las vías de señalización establecidas del TCR inhibió la co-estimulación inducida tanto por Gal-1 como Gal-8, lo cual indica que en realidad estas Gals actúan potenciando las señales débiles de respuestas bordeline del TCR. A pesar de que las células T CD4 constituyen las células blanco del efecto co-estimulador, tanto Gal-1 como Gal-8 tienen que unirse a las CPA y a las células T CD4 de manera simultánea en presencia del antígeno en el modelo de coestimulación. El hecho de que Gal-1 y Gal-8 estimulen las células T en reposo, pero que ejerzan un efecto anti-proliferativo luego de que éstas se activan, propone un rol dual para estas lectinas: por un lado aumentando las respuestas inmunes normales o fisiológicas; y por otro, restringiendo la fase efectora de las respuestas en curso o exacerbadas. Cuando estudiamos la participación de ambas Gals en el inicio de la respuesta inmune in vivo, observamos que una única dosis de Gal-1 o Gal-8, administrada junto con una dosis subóptima de antígeno, fue suficiente para aumentar la respuesta celular T. Además, la administración de Gal-8 en conjunto con el virus de aftosa inactivo (VFA), aumentó la respuesta humoral y de neutralizantes, lo cual se tradujo en un aumento en el porcentaje de protección. En el caso de la inmunización usando Gal-1, no se observaron diferencias en el título de anticuerpos específicos. Por otro lado, encontramos que la respuesta humoral específica contra VFAi inducida por Gal-8 se encontraba acompañada por un aumento en la respuesta celular temprana. En conjunto, nuestros hallazgos demuestran que una única dosis de antígeno VFAi formulado con Gal- 8 desata una respuesta inmune efectiva que protege contra el desafío con virus homólogo,lo cual permite postular esta Gal como potencial adyuvante en algunas preparaciones vacunales. En esta Tesis, describimos por primera vez un efecto activador de Gal-8 sobre células dendríticas (CD), evidenciado por un aumento en la expresión de moléculas coestimuladoras, una mayor capacidad de presentación antigénica, el desarrollo de un fenotipo típico de una CD madura, y una fuerte producción de IL-6. Con respecto a las CD, observamos que éstas también expresan mayor cantidad de Gal-8 luego de su activación. Más aún, las CD provenientes de ratones KO Gal-8-/- expresan menores niveles de CD86 luego de la activación, y además, son menos eficientes a la hora de estimular la proliferación de linfocitos T. En conjunto, el aumento en la expresión de Gal-8, puede constituir un mecanismo endógeno para amplificar el inicio de la respuesta inmune en el sitio de inflamación.Doctor en Biología Molecular y Biotecnologí

Galectin-8 Enhances T cell Response by Promotion of Antigen Internalization and Processing

Galectin-8 (Gal-8) is a mammalian lectin endowed with immunostimulatory ability. In the present work, we demonstrate that Gal-8-glycan interactions on the surface of antigen-presenting cells (APCs) promote antigen binding and internalization, independently from antigen nature. Both Gal-8 and antigen were together internalized and localized in early endosomes. Interestingly, antigen processing by APCs was also accelerated in the presence of Gal-8 as a separate mechanism, distinct from the increased antigen internalization. Moreover, APCs pulsed together with antigen and Gal-8 were able to activate cognate CD4+ T cells more efficiently than those pulsed with antigen alone. This enhanced antigen presentation was still evident in the absence of costimulatory signals and APCs-derived soluble mediators. Therefore, our results provide evidence for as yet unrecognized mechanism by which Gal-8 stimulates the elicitation of the immune response in a lectin-dependent manner, by inducing antigen uptake and processing upon lattice formation at APCs surface.Fil: Prato, Cecilia Arahi. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carabelli, Julieta. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Campetella, Oscar Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Tribulatti, María Virginia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Galectin-8 in the onset of the immune response and inflammation

Galectins (Gals), a family of mammalian lectins, have emerged as key regulators of the immune response, being implicated in several physiologic and pathologic conditions. Lately, there is increasing data regarding the participation of Galectin-8 (Gal-8) in both the adaptive and innate immune responses, as well as its high expression in inflammatory disorders. Here, we focus on the pro- and anti-inflammatory properties of Gal-8 and discuss the potential use of this lectin in order to shape the immune response, according to the context.Fil: Tribulatti, María Virginia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carabelli, Julieta. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Prato, Cecilia Arahi. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Campetella, Oscar Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Purification of Recombinant Galectins Expressed in Bacteria

Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays. For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020).Fil: Prato, Cecilia Arahi. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carabelli, Julieta. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cattaneo, Valentina. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Campetella, Oscar Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Tribulatti, María Virginia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Interleukin-6 signalling mediates Galectin-8 co-stimulatory activity of antigen-specific CD4 T-cell response

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-SL-6 signalling mediates the Gal-8 immune-stimulatory effect.Fil: Carabelli, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Prato, Cecilia Arahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sanmarco, Liliana Maria. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Aoki, Maria del Pilar. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Tribulatti, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Slow progression of pediatric HIV associates with early CD8+ T cell PD-1 expression and a stem-like phenotype

HIV non-progression despite persistent viraemia is rare among antiretroviral therapy (ART)-naïve adults, but relatively common among ART-naïve children. Previous studies indicate that ART-naïve paediatric slow-progressors (PSPs) adopt immune evasion strategies similar to those described in the SIV natural hosts. However, the mechanisms underlying this immunophenotype are not well understood. In a cohort of early-treated infants who underwent analytical treatment interruption (ATI) after 12 months of ART, expression of PD-1 on CD8+ T-cells immediately prior to ATI was the main predictor of slow progression during ATI (r=0.77, p=0.002). PD-1+ CD8+ T-cell frequency was also negatively correlated with CCR5 (r=-0.74, p=0.005) and HLA-DR (r=-0.63, p=0.02) expression on CD4+ T-cells and predicted stronger HIV-specific T-lymphocyte responses. In the CD8+ T-cell compartment of PSPs, we identified an enrichment of stem-like TCF-1+PD-1+ memory cells, whereas paediatric progressors and viraemic adults were populated with a terminally exhausted PD-1+CD39+ population. TCF-1+PD-1+ expression on CD8+ T-cells was associated with higher proliferative activity (r=0.41, p=0.03) and stronger Gag-specific effector functionality. These data prompt the hypothesis that the proliferative burst potential of stem-like HIV-specific cytotoxic cells could be exploited in therapeutic strategies to boost the antiviral response and facilitate remission in early-ART-treated infants with a preserved and non-exhausted T-cell compartment

Slow progression of pediatric HIV associates with early CD8 + T cell PD-1 expression and a stem-like phenotype

HIV nonprogression despite persistent viremia is rare among adults who are naive to antiretroviral therapy (ART) but relatively common among ART-naive children. Previous studies indicate that ART-naive pediatric slow progressors (PSPs) adopt immune evasion strategies similar to those described in natural hosts of SIV. However, the mechanisms underlying this immunophenotype are not well understood. In a cohort of early-treated infants who underwent analytical treatment interruption (ATI) after 12 months of ART, expression of PD-1 on CD8 + T cells immediately before ATI was the main predictor of slow progression during ATI. PD-1 + CD8 + T cell frequency was also negatively correlated with CCR5 and HLA-DR expression on CD4 + T cells and predicted stronger HIV-specific T lymphocyte responses. In the CD8 + T cell compartment of PSPs, we identified an enrichment of stem-like TCF-1 + PD-1 + memory cells, whereas pediatric progressors and viremic adults had a terminally exhausted PD-1 + CD39 + population. TCF-1 + PD-1 + expression on CD8 + T cells was associated with higher proliferative activity and stronger Gag-specific effector functionality. These data prompted the hypothesis that the proliferative burst potential of stem-like HIV-specific cytotoxic cells could be exploited in therapeutic strategies to boost the antiviral response and facilitate remission in infants who received early ART with a preserved and nonexhausted T cell compartment

Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant

Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production and immunogenicity of the recombinant S trimer, suggesting that these two parameters are related. Nevertheless, S-2P still shows some molecular instability and it is produced with low yield. Here we described a novel set of mutations identified by molecular modeling and located in the S2 region of the S-2P that increase its production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development

Galectin‐8 activates dendritic cells and stimulates antigen‐specific immune response elicitation

Galectin‐8 (Gal‐8) is a mammalian β‐galactoside‐binding lectin, endowed with proinflammatory properties. Given its capacity to enhance antigen‐specific immune responses in vivo, we investigated whether Gal‐8 was also able to promote APC activation to sustain T cell activation after priming. Both endogenous [dendritic cells (DCs)] and bone marrow‐derived DCs (BMDCs) treated with exogenous Gal‐8 exhibited a mature phenotype characterized by increased MHC class II (MHCII), CD80, and CD86 surface expression. Moreover, Gal‐8‐treated BMDCs (Gal‐8–BMDCs) stimulated antigen‐specific T cells more efficiently than immature BMDCs (iBMDCs). Proinflammatory cytokines IL‐3, IL‐2, IL‐6, TNF, MCP‐1, and MCP‐5, as well as growth factor G‐CSF, were augmented in Gal‐8–BMDC conditioned media, with IL‐6 as the most prominent. Remarkably, BMDCs from Gal‐8‐deficient mice (Lgals8−/− BMDC) displayed reduced CD86 and IL‐6 expression and an impaired ability to promote antigen‐specific CD4 T cell activation. To test if Gal‐8‐induced activation correlates with the elicitation of an effective immune response, soluble Gal‐8 was coadministrated with antigen during immunization of BALB/cJ mice in the experimental foot‐and‐mouth disease virus (FMDV) model. When a single dose of Gal‐8 was added to the antigen formulation, an increased specific and neutralizing humoral response was developed, sufficient to enhance animal protection upon viral challenge. IL‐6 and IFN‐γ, as well as lymphoproliferative responses, were also incremented in Gal‐8/antigen‐immunized animals only at 48 h after immunization, suggesting that Gal‐8 induces the elicitation of an inflammatory response at an early stage. Taking together, these findings argue in favor of the use of Gal‐8 as an immune‐stimulator molecule to enhance the adaptive immune response.Instituto de VirologíaFil: Carabelli, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Laboratorio de Inmunología Molecular; ArgentinaFil: Quattrocchi, Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: D'Antuono, Alejandra. Instituto de Ciencias y Tecnología “Dr. Cesar Milstein”. Centro de Virología Animal; ArgentinaFil: Zamorano, Patricia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Tribulatti, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Laboratorio de Inmunología Molecular; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Laboratorio de Inmunología Molecular; Argentin

Galectin-8 elicits pro-inflammatory activities in the endothelium

Galectins (Gals), a family of mammalian lectins, play diverse roles under physiological and pathological conditions. Here, we analyzed the tandem-repeat Gal-8 synthesis, secretion and effects on the endothelium physiology. Gal-8M and Gal-8L isoforms were secreted under basal conditions by human microvascular endothelial cells (HMEC-1). However, expression and secretion of the Gal-8M isoform, but not Gal-8L, were increased in response to bacterial lipopolysaccharide (LPS) stimulus and returned to control values after LPS removal. Similarly, cell surface Gal-8 exposure was increased after stimulation with LPS. To evaluate Gal-8 effects on the endothelium physiology, HMEC-1 cells were incubated in the presence of recombinant Gal-8M. Pretreated HMEC-1 cells became proadhesive to human normal platelets, indicating that Gal-8 actually activates endothelial cells. This effect was specific for lectin activity as it was prevented by the simultaneous addition of lactose, but not by sucrose. Endothelial cells also increased their exposition of von Willebrand factor after Gal-8 treatment, which constitutes another feature of cell activation that could be, in turn, responsible for the observed platelet adhesion. Several pro-inflammatory molecules were abundantly produced by Gal-8 stimulated endothelial cells: CXCL1 (GRO-α), GM-CSF, IL-6 and CCL5 (RANTES), and in a lower degree CCL2 (MCP-1), CXCL3 (GRO-γ) and CXCL8 (IL-8). In agreement, Gal-8M induced nuclear factor kappa B phosphorylation. Altogether, these results not only confirm the pro-inflammatory role we have already proposed for Gal-8 in other cellular systems but also suggest that this lectin is orchestrating the interaction between leukocytes, platelets and endothelial cells.Fil: Cattaneo, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Tribulatti, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carabelli, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin