RESUMEN
The therapeutic potential of interleukin (IL)-2 in cancer treatment has been known for decades, yet its widespread adoption in clinical practice remains limited. Recently, chimeric proteins of an anti-PD-1 antibody and suboptimal IL-2 variants were shown to stimulate potent antitumor and antiviral immunity by inducing unique effector CD8+ T cells in mice. A similar subset of cytotoxic T cells is induced by depletion of regulatory T cells (Tregs), suggesting IL-2 sequestration as a major mechanism through which regulatory T cells suppress activated CD8+ T cells. Here, we present our view of how IL-2-based biologicals can boost the antitumor response at a cellular level, and propose that the role of Tregs following such treatments may have been previously overestimated.
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Linfocitos T CD8-positivos , Interleucina-2 , Ratones , Animales , Interleucina-2/uso terapéutico , Interleucina-2/metabolismo , Inmunoterapia , Linfocitos T Citotóxicos , Linfocitos T Reguladores , FenotipoRESUMEN
Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-ß (IL-2Rß):IL-2Rγ heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2Rα (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2Rß and IL-2:IL-2Rγ interactions, but also allosterically lowered the IL-2:IL-2Rα affinity through a "triggered exchange" mechanism favoring IL-2Rα(hi) Treg cells, creating a positive feedback loop for IL-2Rα(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rß interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rß(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.
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Anticuerpos/inmunología , Interleucina-2/metabolismo , Modelos Moleculares , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos/química , Anticuerpos/farmacología , Enfermedades Autoinmunes/inmunología , Unión Competitiva/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-2/química , Interleucina-2/genética , Interleucina-2/inmunología , Ratones , Ratones Endogámicos BALB C , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Acrylamide (AA) a widely used industrial chemical is also formed during food processing by the Maillard reaction, which makes its exposure to humans almost unavoidable. In this study, we used Schizosaccharomyces pombe as a model organism to investigate AA toxicity (10 or 20 mM concentration) in eukaryotes. In S. pombe, AA delays cell growth causes oxidative stress by enhancement of ROS production and triggers excitement of the antioxidant defence system resulting in the division arrest. Aronia fruit contains a variety of health-promoting substances with considerable antioxidant potential. Therefore, Aronia juice supplementation was tested to evaluate its protective effect against AA-derived perturbations of the organism. Cell treatment with several Aronia juice concentrations ranging from 0 to 2% revealed the best protective effect of 1 or 2% Aronia juice solutions. Both chosen Aronia juice concentrations alleviated AA toxicity through the improvement of the antioxidant cell capacity and metabolic activity by their strong ROS scavenging property. Efficiency of Aronia juice cell protection is dose dependent as the 2% solution led to significantly higher cellular defence compared with 1%. Due to the high similarity of biological processes of S. pombe with higher eukaryotes, the protective effect of Aronia juice against AA toxicity might also apply to higher organisms.
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Photinia , Humanos , Photinia/química , Photinia/metabolismo , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Oxidativo , Extractos Vegetales/química , Acrilamidas/farmacologíaRESUMEN
The derivative of protease inhibitor ritonavir (5-methyl-4-oxohexanoic acid ritonavir ester; RD) was recently recognized as a potent P-gp inhibitor and cancerostatic drug inhibiting the proteasome and STAT3 signaling. Therefore, we designed high-molecular-weight HPMA copolymer conjugates with a PAMAM dendrimer core bearing both doxorubicin (Dox) and RD (Star-RD + Dox) to increase the circulation half-life to maximize simultaneous delivery of Dox and RD into the tumor. Star-RD inhibited P-gp activity, potently sensitizing both low- and high-P-gp-expressing cancer cells to the cytostatic and proapoptotic activity of Dox in vitro. Star-RD + Dox possessed higher cytostatic and proapoptotic activities compared to Star-Dox and the equivalent mixture of Star-Dox and Star-RD in vitro. Star-RD + Dox efficiently inhibited STAT3 signaling and induced caspase-3 activation and DNA fragmentation in cancer cells in vivo. Importantly, Star-RD + Dox was found to have superior antitumor activity in terms of tumor growth inhibition and increased survival of mice bearing P-gp-expressing tumors.
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Citostáticos , Neoplasias , Animales , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Ratones , Nanomedicina , Polímeros , Inhibidores de Proteasas/farmacología , RitonavirRESUMEN
Acrylamide (AA), is a chemical with multiple industrial applications, however, it can be found in foods that are rich in carbohydrates. Due to its genotoxic and cytotoxic effects, AA has been classified as a potential carcinogen. With the use of spectrophotometry, ICP-OES, fluorescence spectroscopy, and microscopy cell growth, metabolic activity, apoptosis, ROS production, MDA formation, CAT and SOD activity, ionome balance, and chromosome segregation were determined in Schizosaccharomyces pombe. AA caused growth and metabolic activity retardation, enhanced ROS and MDA production, and modulated antioxidant enzyme activity. This led to damage to the cell homeostasis due to ionome balance disruption. Moreover, AA-induced oxidative stress caused alterations in the cell cycle regulation resulting in chromosome segregation errors, as 4.07% of cells displayed sister chromatid non-disjunction during mitosis. Ascorbic acid (AsA, Vitamin C), a strong natural antioxidant, was used to alleviate the negative impact of AA. Cell pre-treatment with AsA significantly improved AA impaired growth, and antioxidant capacity, and supported ionome balance maintenance mainly due to the promotion of calcium uptake. Chromosome missegregation was reduced to 1.79% (44% improvement) by AsA pre-incubation. Results of our multiapproach analyses suggest that AA-induced oxidative stress is the major cause of alteration to cell homeostasis and cell cycle regulation.
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Ácido Ascórbico , Schizosaccharomyces , Acrilamida/toxicidad , Antioxidantes/metabolismo , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Mitosis , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Schizosaccharomyces/metabolismoRESUMEN
In our study we investigated the effect of different nickel (NiSO4·6H2O) (Ni) concentrations on cell division, cellular morphology and ionome homeostasis of the eukaryotic model organism Schizosaccharomyces pombe. Target of rapamycin (TOR) protein kinase is one of the key regulators of cell growth under different environmental stresses. We analyzed the effect of Ni on cell strains lacking the Tor1 signaling pathway utilizing light-absorbance spectroscopy, visualization, microscopy and inductively coupled plasma optical emission spectroscopy. Interestingly, our findings revealed that Ni mediated cell growth alterations are noticeably lower in Tor1 deficient cells. Greater size of Tor1 depleted cells reached similar quantitative parameters to wild type cells upon incubation with 400 µM Ni. Differences of ion levels among the two tested yeast strains were detected even before Ni addition. Addition of high concentration (1 mM) of the heavy metal, representing acute contamination, caused considerable changes in the ionome of both strains. Strikingly, Tor1 deficient cells displayed largely reduced Ni content after treatment compared to wild type controls (644.1 ± 49 vs. 2096.8 ± 75 µg/g), suggesting its significant role in Ni trafficking. Together our results predict yet undefined role for the Tor1 signaling in metal uptake and/or metabolism.
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Regulación Enzimológica de la Expresión Génica , Níquel/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Regulación hacia Abajo , Activación Enzimática , Regulación Fúngica de la Expresión Génica , Cinética , Níquel/química , Proteínas Quinasas/química , Proteínas Quinasas/genética , Schizosaccharomyces/química , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Supraphysiological levels of IL-7 induce increase counts of pre-B cells, naive T cells and memory phenotype CD8+ T cells. Immunocomplexes of IL-7 and αIL-7 mAb M25 (IL-7/M25) were described as IL-7 superagonist in vivo. Thus, treatment of mice with IL-7/M25 remarkably increases the size of the T cell pool. We decided to use IL-7/M25 in order to expand the T cell population prior to the administration of αCTLA-4 and αPD-1 mAbs in tumor-bearing mice and in turn boost the immunotherapy based on a combination of CTLA-4 and PD-1 blockage. We found that just four doses of IL-7/M25 increased the absolute numbers of splenocytes approximately fivefold and significantly shifted the CD4+:CD8+ T cell ratio in favor of CD8+ T cells. There was also a substantive increase in relative counts of memory phenotype CD8+ T cells (approximately threefold) within CD8+ T cells but a significant decrease (approximately 30%) in relative counts of Treg cells within CD4+ T cells. All these data suggest that IL-7/M25 offer a suitable approach to potentiate tumor immunotherapy through CTLA-4 and PD-1 blockage. Unexpectedly, IL-7/M25 significantly abrogated the antitumor activity of αCTLA-4 plus αPD-1 mAbs in the following mouse tumor models: MC-38 and CT26 colon carcinoma and B16F10 melanoma. This paradoxical effect of IL-7/M25 on the antitumor activity of CTLA-4 and PD-1 blockage was not mediated via either increased levels of IL-10 or TGF-ß in the sera or increased counts of IL-10-producing B or T cells in the spleen of mice injected with IL-7/M25. Thus, our work shows that caution should be exercised when combining two immunotherapy approaches together.
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Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/inmunología , Interleucina-7/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antineoplásicos/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoterapia/métodos , Interleucina-10/inmunología , Masculino , Melanoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns. Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). Calreticulin exposure not only correlated with enhanced T-cell-dependent antitumor immunity in this setting but also affected the number of circulating natural killer (NK) cells upon restoration of normal hematopoiesis. Here, we report that calreticulin exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of calreticulin to stimulate NK-cells relies on CD11c+CD14high cells that, upon exposure to CRT, express higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR) and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferon. This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed calreticulin favors NK-cell activation in AML patients.
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Calreticulina , Leucemia Mieloide Aguda , Animales , Calreticulina/genética , Calreticulina/metabolismo , Citotoxicidad Inmunológica , Humanos , Interleucina-15 , Células Asesinas Naturales , Leucemia Mieloide Aguda/terapia , Activación de Linfocitos , RatonesRESUMEN
IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rß, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.
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Anticuerpos/metabolismo , Enfermedades Autoinmunes/inmunología , Colitis/inmunología , Citocinas/metabolismo , Inmunoterapia/métodos , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos/genética , Enfermedades Autoinmunes/terapia , Proliferación Celular , Células Cultivadas , Colitis/terapia , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Humanos , Activación de Linfocitos , Ratones , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Cadmium has no known physiological function in the body; however, its adverse effects are associated with cancer and many types of organ system damage. Although much has been shown about Cd toxicity, the underlying mechanisms of its responses to the organism remain unclear. In this study, the role of Tor1, a catalytic subunit of the target of rapamycin complex 2 (TORC2), in Cd-mediated effects on cell proliferation, the antioxidant system, morphology, and ionome balance was investigated in the eukaryotic model organism Schizosaccharomyces pombe. Surprisingly, spectrophotometric and biochemical analyses revealed that the growth rate conditions and antioxidant defense mechanisms are considerably better in cells lacking the Tor1 signaling. The malondialdehyde (MDA) content of Tor1-deficient cells upon Cd treatment represents approximately half of the wild-type content. The microscopic determination of the cell morphological parameters indicates the role for Tor1 in cell shape maintenance. The ion content, determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), showed that the Cd uptake potency was markedly lower in Tor1-depleted compared to wild-type cells. Conclusively, we show that the cadmium-mediated cell impairments in the fission yeast significantly depend on the Tor1 signaling. Additionally, the data presented here suggest the yet-undefined role of Tor1 in the transport of ions.
Asunto(s)
Cadmio/toxicidad , Proteínas Quinasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/efectos de los fármacos , Cadmio/administración & dosificación , Relación Dosis-Respuesta a Droga , Regulación Fúngica de la Expresión Génica , Homeostasis/efectos de los fármacos , Iones/metabolismo , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas Quinasas/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMß2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b- cells. The nonhemolytic AC+ Hly- bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly- mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b- cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.
Asunto(s)
Toxina de Adenilato Ciclasa/metabolismo , Bordetella pertussis/patogenicidad , AMP Cíclico/metabolismo , Proteínas Hemolisinas/metabolismo , Antígeno de Macrófago-1/metabolismo , Tos Ferina/microbiología , Animales , Antígeno CD11b/metabolismo , Membrana Celular/metabolismo , Células Dendríticas/inmunología , Femenino , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fagocitos/inmunología , Linfocitos T/inmunología , VirulenciaRESUMEN
The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.
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Toxina de Adenilato Ciclasa/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Activación de Linfocitos , Proteínas Citotóxicas Formadoras de Poros/inmunología , Dominios Proteicos/inmunología , Toxina de Adenilato Ciclasa/genética , Adyuvantes Inmunológicos/genética , Animales , Vacunas contra el Cáncer/inmunología , Diferenciación Celular , Permeabilidad de la Membrana Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/microbiología , Transporte Iónico , Ratones , Ratones Endogámicos C57BL , Proteínas Citotóxicas Formadoras de Poros/genética , Dominios Proteicos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The study evaluates the impact of two metal oxide nanoparticles: copper oxide (CuO) and zinc oxide (ZnO) on the growth and physiology of Raphanus sativus L. (radish) under salinity stress. Fifteen days old seedlings of R. sativus were subjected to different concentrations of salt stress (0 mM, 150 mM, and 300 mM NaCl) alone and in interaction with 100 mgL-1 metal oxide nanoparticle treatments (CuO and ZnO NPs via foliar spray) for 15 days. The results confirmed the severe effects of salinity stress on the growth and physiology of radish plants by decreasing nutrient uptake, leaf area, and photosystems photochemistry and by increasing proline accumulation, anthocyanin, flavonoids content, and antioxidant enzyme activities which is directly linked to increased oxidative stress. The foliar application of CuO and ZnO NPs alleviated the adverse effects of salt stress on radish plants, as indicated by improving these attributes. Foliar spray of ZnO NPs was found efficient in improving the leaf area, photosynthetic electron transport rate, the PSII quantum yield, proton conductance and mineral content in radish plants under NaCl stress. Besides, ZnO NPs decreased the NaCl-induced oxidative stress by declining proline, anthocyanin, and flavonoids contents and enzymatic activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and guaiacol peroxidase (GOPX). Thus, our study revealed that ZnO NPs are more effective and have beneficial effects over CuO NPs in promoting growth and reducing the adverse effects of NaCl stress in radish plants.
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Nanopartículas del Metal , Nanopartículas , Raphanus , Óxido de Zinc , Óxido de Zinc/farmacología , Raphanus/metabolismo , Cobre/farmacología , Antocianinas , Cloruro de Sodio/farmacología , Fotosíntesis , Antioxidantes/metabolismo , Estrés Salino , Prolina/metabolismoRESUMEN
Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins' limitations as drugs. The interleukin-2 (IL-2) cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory regulatory T cells, toxicity at high doses, and short serum half-life. One approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards immune effector cell activation. Although this strategy shows potential in preclinical models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns regarding complex stability. Here, we introduced a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine towards immune effector cells. We optimized IC construction and engineered the cytokine/antibody affinity to improve immune bias. We demonstrated that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2, both alone and combined with immune checkpoint inhibitors. Moreover, therapeutic efficacy was observed without inducing toxicity. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.
RESUMEN
PURPOSE: Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICIs) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. EXPERIMENTAL DESIGN: We harnessed a variety of transcriptomic, spatial and functional assays to characterize the differential impact of neo-adjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to NACT-naïve HGSOC samples from 5 independent patient cohorts. RESULTS: We found neo-adjuvant chemotherapy (NACT)-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD-1+ CD8+ T cells over their ICI-insensitive TIM-3+PD-1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD-1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. CONCLUSION: Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC.
RESUMEN
Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neoplasias Ováricas , Estructuras Linfoides Terciarias , Humanos , Femenino , Linfocitos T CD8-positivos , Neoplasias Ováricas/patología , Linfocitos Infiltrantes de Tumor , Fenotipo , Microambiente TumoralRESUMEN
The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.
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Acrilamidas/farmacología , Materiales Biocompatibles/farmacología , Citostáticos/farmacología , Leucemia/tratamiento farmacológico , Polímeros/farmacología , Acrilamidas/síntesis química , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/química , Materiales Biocompatibles/síntesis química , Línea Celular Tumoral , Proliferación Celular , Citostáticos/química , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Metacrilatos/química , Ratones , Ratones Endogámicos BALB C , Polímeros/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Loss of muscle mass occurs rapidly during critical illness and negatively affects quality of life. The incidence of clinically significant muscle wasting in critically ill patients is unclear. This study aimed to assess the incidence of and identify predictors for clinically significant loss of muscle mass in this patient population. This was a single-center observational study. We used ultrasound to determine the rectus femoris cross-sectional area (RFcsa) on the first and seventh day of ICU stay. The primary outcome was the incidence of significant muscle wasting. We used a logistic regression model to determine significant predictors for muscle wasting. Ultrasound measurements were completed in 104 patients. Sixty-two of these patients (59.6%) showed ≥ 10% decreases in RFcsa. We did not identify any predictor for significant muscle wasting, however, age was of borderline significance (p = 0.0528). The 28-day mortality rate was higher in patients with significant wasting, but this difference was not statistically significant (30.6% versus 16.7%; p = 0.165). Clinically significant muscle wasting was frequent in our cohort of patients. Patient age was identified as a predictor of borderline significance for muscle wasting. The results could be used to plan future studies on this topic.Trial registration: ClinicalTrials.gov NCT03865095, date of registration: 06/03/2019.
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Enfermedad Crítica , Calidad de Vida , Humanos , Estudios Prospectivos , Incidencia , Unidades de Cuidados Intensivos , Atrofia Muscular/diagnóstico por imagen , Atrofia Muscular/epidemiología , Atrofia Muscular/etiología , Músculo Cuádriceps/diagnóstico por imagen , UltrasonografíaRESUMEN
The rate of global environmental change is unprecedented, with climate change causing an increase in the oscillation and intensification of various abiotic stress factors that have negative impacts on crop production. This issue has become an alarming global concern, especially for countries already facing the threat of food insecurity. Abiotic stressors, such as drought, salinity, extreme temperatures, and metal (nanoparticle) toxicities, are recognized as major constraints in agriculture, and are closely associated with the crop yield penalty and losses in food supply. In order to combat abiotic stress, it is important to understand how plant organs adapt to changing conditions, as this can help produce more stress-resistant or stress-tolerant plants. The investigation of plant tissue ultrastructure and subcellular components can provide valuable insights into plant responses to abiotic stress-related stimuli. In particular, the columella cells (statocytes) of the root cap exhibit a unique architecture that is easily recognizable under a transmission electron microscope, making them a useful experimental model for ultrastructural observations. In combination with the assessment of plant oxidative/antioxidative status, both approaches can shed more light on the cellular and molecular mechanisms involved in plant adaptation to environmental cues. This review summarizes life-threatening factors of the changing environment that lead to stress-related damage to plants, with an emphasis on their subcellular components. Additionally, selected plant responses to such conditions in the context of their ability to adapt and survive in a challenging environment are also described.