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Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24â¯h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4â¯h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.
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BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.
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Interferón gamma , Sepsis , Humanos , Interferón gamma/metabolismo , Inmunoadsorbentes/uso terapéutico , Estudios Prospectivos , BiomarcadoresRESUMEN
Uropathogenic E. coli (UPEC) is a primary organism responsible for urinary tract infections and a common cause of sepsis. Microbially experienced laboratory mice, generated by cohousing with pet store mice, exhibit increased morbidity and mortality to polymicrobial sepsis or lipopolysaccharide challenge. By contrast, cohoused mice display significant resistance, compared with specific pathogen-free mice, to a monomicrobial sepsis model using UPEC. CD115+ monocytes mediate protection in the cohoused mice, as depletion of these cells leads to increased mortality and UPEC pathogen burden. Further study of the cohoused mice reveals increased TNF-α production by monocytes, a skewing toward Ly6ChiCD115+ "classical" monocytes, and enhanced egress of Ly6ChiCD115+ monocytes from the bone marrow. Analysis of cohoused bone marrow also finds increased frequency and number of myeloid multipotent progenitor cells. These results show that a history of microbial exposure impacts innate immunity in mice, which can have important implications for the preclinical study of sepsis.
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Infecciones por Escherichia coli , Sepsis , Infecciones Urinarias , Escherichia coli Uropatógena , Ratones , Animales , Monocitos , Escherichia coli , Inmunidad Innata , Proteínas Tirosina Quinasas ReceptorasRESUMEN
BACKGROUND: Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. METHODS: An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. RESULTS: Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. CONCLUSIONS: A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients.
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Microbial experience fundamentally shapes immunity, particularly during the perinatal period when the immune system is underdeveloped, and novel microbial encounters are common. Most animal models are raised in specific pathogen-free (SPF) conditions with relatively uniform microbial communities. How SPF housing conditions alter early-life immune development relative to natural microbial exposure (NME) has not been thoroughly investigated. In this article, we compare immune development in SPF-raised mice with mice born from immunologically experienced mothers in microbially diverse environments. NME induced broad immune cell expansion, including naive cells, suggesting mechanisms besides activation-induced proliferation contribute to the increase in immune cell numbers. We found NME conditions also expanded immune cell progenitor cell populations in the bone marrow, suggesting microbial experience enhances immune development at the earliest stages of immune cell differentiation. Multiple immune functions characteristically impaired in infants were also enhanced by NME, including T cell memory and Th1 polarization, B cell class switching and Ab production, proinflammatory cytokine expression, and bacterial clearance after Listeria monocytogenes challenge. Collectively, our studies reveal numerous impairments in immune development in SPF conditions relative to natural immune development.
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Citocinas , Listeria monocytogenes , Animales , Ratones , Citocinas/metabolismo , Médula Ósea/metabolismo , Linfocitos B , Células Madre/metabolismo , Ratones Endogámicos C57BLRESUMEN
Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.
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Interleucina-27 , Humanos , Ratones , Animales , Interleucina-27/metabolismo , Linfocitos T CD8-positivos , Antígenos CD40 , Diferenciación Celular , Células DendríticasRESUMEN
Lung group 2 innate lymphoid cells (ILC2s) control the nature of immune responses to airway allergens. Some microbial products, including those that stimulate interferons, block ILC2 activation, but whether this occurs after natural infections or causes durable ILC2 inhibition is unclear. In the present study, we cohoused laboratory and pet store mice as a model of physiological microbial exposure. Laboratory mice cohoused for 2 weeks had impaired ILC2 responses and reduced lung eosinophilia to intranasal allergens, whereas these responses were restored in mice cohoused for ≥2 months. ILC2 inhibition at 2 weeks correlated with increased interferon receptor signaling, which waned by 2 months of cohousing. Reinduction of interferons in 2-month cohoused mice blocked ILC2 activation. These findings suggest that ILC2s respond dynamically to environmental cues and that microbial exposures do not control long-term desensitization of innate type 2 responses to allergens.
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Alérgenos , Inmunidad Innata , Ratones , Animales , Linfocitos , Citocinas , Pulmón , Interferones , Interleucina-33RESUMEN
Toll-like receptors (TLRs) 7 and 8 are key targets in the development of immunomodulatory drugs for treating infectious disease, cancer, and autoimmune disorders. These receptors can adopt both agonist and antagonist binding conformations that switch the receptor signal on or off to the downstream production of cytokines. In this study, we examined the effect of simple isomeric substitutions to the C2-butyl group of two imidazoquinoline agonists and evaluated the activity of these analogs using both TLR7 and TLR8 reporter cells and cytokine induction assays. Results are presented showing the C2-isobutyl and C2-cyclopropylmethyl isomers are both mixed TLR7/8 competitive antagonists of the parent agonist [4-Amino-1-(4-(aminomethyl)benzyl)-2-butyl-7-methoxycarbonyl-1H-imidazo[4,5-c]quinoline], indicating the conformation of the dimeric receptor complex is highly sensitive to steric perturbations to the ligand binding pocket. This observation is consistent with prior work demonstrating TLR7 and TLR8 activity is directly correlated to C2-alkyl substitutions that project into a hydrophobic pocket at the dimer interface of the receptor. The close structural relationship of the agonist/antagonist pairs identified here highlights the importance of this pocket in tipping the balance between the agonist and antagonist binding states of the receptor which may have significant ramifications to the design of imidazoquinoline-based immunomodulatory agents.
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Imidazoles/farmacología , Quinolinas/farmacología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
Patients who survive sepsis display prolonged immune dysfunction and heightened risk of secondary infection. CD4 T cells support a variety of cells required for protective immunity, and perturbations to the CD4 T cell compartment can decrease overall immune system fitness. Using the cecal ligation and puncture (CLP) mouse model of sepsis, we investigated the impact of sepsis on endogenous Ag-specific memory CD4 T cells generated in C57BL/6 (B6) mice infected with attenuated Listeria monocytogenes (Lm) expressing the I-Ab-restricted 2W1S epitope (Lm-2W). The number of 2W1S-specific memory CD4 T cells was significantly reduced on day 2 after sepsis induction, but recovered by day 14. In contrast to the transient numerical change, the 2W1S-specific memory CD4 T cells displayed prolonged functional impairment after sepsis, evidenced by a reduced recall response (proliferation and effector cytokine production) after restimulation with cognate Ag. To define the extent to which the observed functional impairments in the memory CD4 T cells impacts protection to secondary infection, B6 mice were infected with attenuated Salmonella enterica-2W (Se-2W) 30 days before sham or CLP surgery, and then challenged with virulent Se-2W after surgery. Pathogen burden was significantly higher in the CLP-treated mice compared to shams. Similar reductions in functional capacity and protection were noted for the endogenous OVA323-specific memory CD4 T cell population in sepsis survivors upon Lm-OVA challenge. Our data collectively show CLP-induced sepsis alters the number and function of Ag-specific memory CD4 T cells, which contributes (in part) to the characteristic long-lasting immunoparalysis seen after sepsis.
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Linfocitos T CD4-Positivos/inmunología , Coinfección/inmunología , Inmunidad Celular , Memoria Inmunológica , Sepsis/inmunología , Animales , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Ciego/microbiología , Ciego/cirugía , Proliferación Celular , Coinfección/metabolismo , Coinfección/microbiología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Ligadura , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Listeriosis/inmunología , Listeriosis/metabolismo , Listeriosis/microbiología , Activación de Linfocitos , Ratones Endogámicos C57BL , Punciones , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Salmonella enterica/inmunología , Salmonella enterica/patogenicidad , Sepsis/metabolismo , Sepsis/microbiologíaRESUMEN
Activated natural killer (NK) cells can kill malignant tumor cells via granule exocytosis and secretion of IFN-γ, a key regulator of the TH1 response. Thus, mobilization of NK cells can augment cancer immunotherapy, particularly when mediated through antibody-dependent cellular cytotoxicity (ADCC). Stimulation of toll-like receptor (TLR)7/8 activity in dendritic cells promotes pro-inflammatory cytokine secretion and costimulatory molecule upregulation, both of which can potentiate NK cell activation. However, currently available TLR7/8 agonists exhibit unfavorable pharmacokinetics, limiting their in vivo efficacy. To enable efficient delivery to antigen-presenting cells, we encapsulated a novel imidazoquinoline-based TLR7/8 agonist in pH-responsive polymeric NPs. Enhanced costimulatory molecule expression on dendritic cells and a stronger pro-inflammatory cytokine response were observed with a NP-encapsulated agonist, compared to that with the soluble form. Treatment with NP-encapsulated agonists resulted in stronger in vivo cytotoxicity and prolonged activation of NK cells compared to that with a soluble agonist. In addition, TLR7/8 agonist-loaded NPs potentiated stronger NK cell degranulation, which resulted in enhanced in vitro and in vivo ADCC mediated by the epidermal growth factor receptor-targeting antibody cetuximab. TLR7/8 agonist-loaded NP treatment significantly enhanced the antitumor efficacy of cetuximab and an anti-HER2/neu antibody in mouse tumor models. Collectively, our data show that a pH-responsive NP-encapsulating TLR7/8 agonist could be used as a potent immunostimulatory adjuvant for antibody-based cancer immunotherapy by promoting NK cell activation.
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Inmunoterapia/métodos , Células Asesinas Naturales/metabolismo , Nanopartículas/química , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Células A549 , Animales , Células Dendríticas/metabolismo , Citometría de Flujo , Humanos , Imiquimod/química , Células Asesinas Naturales/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanomedicina/métodosRESUMEN
Microbial exposures can define an individual's basal immune state. Cohousing specific pathogen-free (SPF) mice with pet store mice, which harbor numerous infectious microbes, results in global changes to the immune system, including increased circulating phagocytes and elevated inflammatory cytokines. How these differences in the basal immune state influence the acute response to systemic infection is unclear. Cohoused mice exhibit enhanced protection from virulent Listeria monocytogenes (LM) infection, but increased morbidity and mortality to polymicrobial sepsis. Cohoused mice have more TLR2+ and TLR4+ phagocytes, enhancing recognition of microbes through pattern-recognition receptors. However, the response to a TLR2 ligand is muted in cohoused mice, whereas the response to a TLR4 ligand is greatly amplified, suggesting a basis for the distinct response to Listeria monocytogenes and sepsis. Our data illustrate how microbial exposure can enhance the immune response to unrelated challenges but also increase the risk of immunopathology from a severe cytokine storm.
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Citocinas/metabolismo , Inmunidad Innata/inmunología , Inflamación/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Femenino , Inflamación/metabolismo , Inflamación/patología , Listeriosis/metabolismo , Listeriosis/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Fagocitos/inmunología , Fagocitos/metabolismo , Fagocitos/patología , Sepsis/inmunología , Sepsis/metabolismo , Sepsis/patología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genéticaRESUMEN
Cancer vaccines composed of tumor-associated antigens (TAAs) and toll-like receptor (TLR) agonists have shown promising antitumor efficacy in preclinical studies by generating antigen-specific CD8 T cells, but translation of cancer vaccines to the clinic has been limited due to variables responses and development of resistance. The tumor microenvironment deploys various immune escape mechanisms that neutralize CD8 T cell-mediated tumor rejection. Therefore, we hypothesized that modulation of the tumor microenvironment can augment CD8 T cell activation and enhance therapeutic efficacy of cancer vaccines. To accomplish this, we aimed to eliminate immune suppressive cells and block their inhibitory signaling. Combination of the tyrosine kinase inhibitor (TKI) sunitinib with a nanoparticle-based cancer vaccine (nanovaccine) resulted in the reduction of immune-suppressive myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs). Blockade of programmed death-ligand 1 (PD-L1) using anti-PD-L1 antibody was used to reduce CD8 T cell exhaustion. Combination of nanovaccine+sunitinib+PD-L1 antibody treatment reduced PD-L1high M2 macrophages and MDSCs and upregulated activation of CD8 T cells in the tumor. Nanovaccine+sunitinib+PD-L1 antibody treatment also stimulated antigen-specific CD8 T cell response, which led to improved therapeutic efficacy in MB49 and B16F10 murine tumor models. These results suggest that modulation of tumor microenvironment using sunitinib and PD-L1 blockade can significantly enhance the antitumor efficacy of cancer nanovaccine.
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Anticuerpos/uso terapéutico , Antígeno B7-H1/inmunología , Vacunas contra el Cáncer/uso terapéutico , Glicoproteínas de Membrana/agonistas , Neoplasias/terapia , Sunitinib/uso terapéutico , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Animales , Antígenos de Neoplasias/inmunología , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Femenino , Interleucina-10/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Linfocitos T Reguladores/inmunología , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , VacunaciónRESUMEN
Immunosuppression is one hallmark of sepsis, decreasing the host response to the primary septic pathogens and/or secondary nosocomial infections. CD4 T cells and B cells are among the array of immune cells that experience reductions in number and function during sepsis. "Help" from follicular helper (Tfh) CD4 T cells to B cells is needed for productive and protective humoral immunity, but there is a paucity of data defining the effect of sepsis on a primary CD4 T cell-dependent B cell response. Using the cecal ligation and puncture (CLP) mouse model of sepsis induction, we observed reduced antibody production in mice challenged with influenza A virus or TNP-KLH in alum early (2 days) and late (30 days) after CLP surgery compared to mice subjected to sham surgery. To better understand how these CD4 T cell-dependent B cell responses were altered by a septic event, we immunized mice with a Complete Freund's Adjuvant emulsion containing the MHC II-restricted peptide 2W1S56-68 coupled to the fluorochrome phycoerythrin (PE). Immunization with 2W1S-PE/CFA results in T cell-dependent B cell activation, giving us the ability to track defined populations of antigen-specific CD4 T cells and B cells responding to the same immunogen in the same mouse. Compared to sham mice, differentiation and class switching in PE-specific B cells were blunted in mice subjected to CLP surgery. Similarly, mice subjected to CLP had reduced expansion of 2W1S-specific T cells and Tfh differentiation after immunization. Our data suggest CLP-induced sepsis impacts humoral immunity by affecting the number and function of both antigen-specific B cells and CD4 Tfh cells, further defining the period of chronic immunoparalysis after sepsis induction.
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Linfocitos B/inmunología , Coinfección/inmunología , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/inmunología , Sepsis/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Anticuerpos/metabolismo , Antígenos Bacterianos/inmunología , Ciego/cirugía , Diferenciación Celular , Células Cultivadas , Enfermedad Crónica , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Tolerancia Inmunológica , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BLRESUMEN
Synthetic imidazoquinoline-based toll-like receptor (TLR) 7/8 bi-specific agonists are promising vaccine adjuvants that can induce maturation of dendritic cells (DCs) and activate them to secrete pro-inflammatory cytokines. However, in vivo efficacy of these small molecule agonists is often hampered by their fast clearance from the injection site, limiting their use to topical treatments. In this study, we investigated the use of acidic pH-responsive poly(lactide-co-glycolide) (PLGA) nanoparticles for endo-lysosome specific release of 522, a novel TLR7/8 agonist. Bicarbonate salt was incorporated into the new formulation to generate carbon dioxide (CO2) gas at acidic pH, which can disrupt the polymer shell to rapidly release the payload. Compared to conventional PLGA nanoparticles, the pH responsive formulation resulted in 33-fold higher loading of 522. The new formulation demonstrated acid-responsive CO2 gas generation and drug release. The acid-responsive formulation increased the in vitro expression of co-stimulatory molecules on DCs and improved antigen-presentation via MHC I, both of which are essential for CD8 T cell priming. In vivo studies showed that the pH-responsive formulation elicited stronger antigen-specific CD8 T cell and natural killer (NK) cell responses than conventional PLGA nanoparticles, resulting in enhanced anticancer efficacy in a murine melanoma tumor model. Our results suggest that acidic-pH responsive, gas-generating nanoparticles are an efficient TLR7/8 agonist delivery platform for cancer immunotherapy.
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Portadores de Fármacos/química , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Animales , Presentación de Antígeno , Células de la Médula Ósea/citología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Dióxido de Carbono/metabolismo , Línea Celular Tumoral , Microscopía por Crioelectrón , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Concentración de Iones de Hidrógeno , Inmunoterapia , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Neoplasias/terapia , Ovalbúmina/inmunologíaRESUMEN
Various malignancies are reproducibly cured in mouse models, but most cancer immunotherapies show objective responses in a fraction of treated patients. One reason for this disconnect may be the use of young, lean mice lacking immune-altering comorbidities present in cancer patients. Although many cancer patients are overweight or obese, the effect of obesity on antitumor immunity is understudied in preclinical tumor models. We examined the effect of obesity on two immunotherapeutic models: systemic anti-CTLA-4 mAb and intratumoral delivery of a TRAIL-encoding adenovirus plus CpG. Both therapies were effective in lean mice, but neither provided a survival benefit to diet-induced obese BALB/c mice. Interestingly, tumor-bearing leptin-deficient (ob/ob) obese BALB/c mice did respond to treatment. Moreover, reducing systemic leptin with soluble leptin receptor:Fc restored the antitumor response in diet-induced obese mice. These data demonstrate the potential of targeting leptin to improve tumor immunotherapy when immune-modulating comorbidities are present.
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Adenocarcinoma/metabolismo , Envejecimiento/fisiología , Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia/métodos , Neoplasias Renales/metabolismo , Leptina/metabolismo , Obesidad/metabolismo , Adenocarcinoma/terapia , Adenoviridae/genética , Animales , Antígeno CTLA-4/inmunología , Línea Celular Tumoral , Dieta , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad , Neoplasias Renales/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Obesidad/terapia , Oligodesoxirribonucleótidos/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Cytotoxic T lymphocytes (CTLs) play a major role in cancer immunotherapy because of their ability to directly kill tumor cells and secrete tumor suppressive cytokines. Anticancer vaccines aim to provoke tumor-specific CTL responses, which require activation of antigen presenting cells (APCs) including dendritic cells (DCs) and macrophages. Therefore, a potent immunostimulatory adjuvant capable of activating APCs is an essential component of anticancer vaccines. In this study, we introduce novel TLR 7/8 bi-specific agonists that significantly enhance cytokine secretion compared to TLR7 mono-selective compounds. Encapsulation of these TLR 7/8 agonists in poly(lactide-co-glycolide) (PLGA) nanoparticles increased the co-stimulatory molecule expression and antigen presentation via MHC I by DCs compared to the soluble agonist. When administered subcutaneously, these nanoparticles migrated to draining lymph node and triggered DC activation and expansion. This lead to expansion of antigen-specific CD8 T cells and enhanced CTL response, which resulted in significant prophylactic and therapeutic efficacy in melanoma, bladder and renal cell carcinoma tumor models. Importantly, our studies demonstrate significant reductions in systemic metastasis with the nanoparticle vaccine. Our results suggest novel TLR 7/8 agonist-encapsulated nanoparticles are potent immunostimulatory adjuvants for cancer immunotherapy.
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Portadores de Fármacos , Imidazoles/farmacología , Nanocápsulas , Poliglactina 910 , Quinolinas/farmacología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Adyuvantes Inmunológicos/farmacología , Animales , Antineoplásicos/farmacología , Portadores de Fármacos/química , Femenino , Humanos , Inmunoterapia/métodos , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Linfocitos T Citotóxicos/efectos de los fármacosRESUMEN
A series of N1-modified imidazoquinolines were synthesized and screened for Toll-like receptors (TLR) 7 and 8 activities to identify recognition elements that confer high affinity binding and selectivity. These receptors are key targets in the development of immunomodulatory agents that signal the NF-κB mediated transcription of pro-inflammatory chemokines and cytokines. Results are presented showing both TLR7/8 activations are highly correlated to N1-substitution, with TLR8 selectivity achieved through inclusion of an ethyl-, propyl-, or butylamino group at this position. While the structure-activity relationship analysis indicates TLR7 activity is less sensitive to N1-modification, extension of the aminoalkyl chain length to pentyl and p-methylbenzyl elicited high affinity TLR7 binding. Cytokine profiles are also reported that show the pure TLR8 agonist [4-amino-2-butyl-1-(2-aminoethyl)-7-methoxycarbonyl-1H-imidazo[4,5-c]quinoline] induces higher levels of IL-1ß, IL-12, and IFNγ when compared with TLR7 selective or mixed TLR7/8 agonists. The results are consistent with previous work suggesting TLR8 agonists are Th1 polarizing and may help promote cell-mediated immunity.
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Patients surviving acute stages of sepsis often display impaired adaptive-immune responses. Using the cecal ligation and puncture model, we demonstrated that sepsis leads to substantial and long-lasting changes in the naive CD8 T cell repertoire, affecting the capacity of the host to respond to new infections. However, the identity of CD8 T cell-extrinsic factor(s) and mechanism(s) that contribute to impaired CD8 T cell responses after sepsis is unknown. Priming of naive CD8 T cells is critically dependent on the ability of dendritic cells (DCs) to provide Ag, costimulation, and inflammatory signal 3 cytokines; therefore, the sepsis-induced changes in the DC compartment might represent a contributing factor leading to diminished CD8 T cell immunity in septic hosts. In a direct test of this hypothesis, we show that, in addition to numerical decline, sepsis leads to functional impairments in DCs, diminishing their capacity to produce cytokines upon TLR stimulation in vitro or postinfection in vivo. Importantly, we demonstrated a direct link between DC dysfunction and impairments in CD8 T cell immunity after sepsis by directly targeting Ag to DCs. Finally, postsepsis Flt3 ligand treatment increased the number of DCs and improved DC function, including the ability to sense inflammation and produce IL-12, leading to improved primary CD8 T cell responses to newly encountered Ags. Thus, sepsis-induced numerical and functional loss of DCs contributes to the observed defects in CD8 T cell immunity, and therapeutic approaches designed to improve the status of the DC compartment after sepsis might facilitate the recovery of CD8 T cell immunity.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Sepsis/inmunología , Animales , Antígenos/genética , Antígenos/inmunología , Linfocitos T CD8-positivos/patología , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/patología , Ratones , Ratones Transgénicos , Sepsis/genética , Sepsis/patologíaRESUMEN
Transient lymphopenia is one hallmark of sepsis, and emergent data indicate the CD4 T cell compartment in sepsis survivors is numerically and functionally altered (when examined at the Ag-specific level) compared with nonseptic control subjects. Previous data from our laboratory demonstrated Ag-independent, lymphopenia-induced homeostatic proliferation to be a contributing mechanism by which CD4 T cells numerically recover in sepsis survivors. However, we reasoned it is also formally possible that some CD4 T cells respond directly to Ag expressed by gut-resident microbes released during polymicrobial sepsis. The effect of gut microbiome leakage on CD4 T cells is currently unknown. In this study, we explored the number and function of endogenous CD4 T cells specific for segmented filamentous bacterium (SFB) after cecal ligation and puncture (CLP)-induced sepsis using mice that either contained or lacked SFB as a normal gut-resident microbe. Interestingly, SFB-specific CD4 T cells underwent Ag-driven proliferation in CLP-treated SFB(+), but not in SFB(-), mice. Moreover, CLP-treated SFB(+) mice showed resistance to secondary lethal infection with recombinant SFB Ag-expressing virulent Listeria (but not wild-type virulent Listeria), suggesting the CLP-induced polymicrobial sepsis primed for a protective response by the SFB-specific CD4 T cells. Thus, our data demonstrate that the numerical recovery and functional responsiveness of Ag-specific CD4 T cells in sepsis survivors is, in part, modulated by the intestinal barrier's health discreetly defined by individual bacterial populations of the host's microbiome.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Microbioma Gastrointestinal , Sepsis/inmunología , Sepsis/microbiología , Animales , Ciego/cirugía , Citometría de Flujo , Microbioma Gastrointestinal/inmunología , Intestinos/microbiología , Listeria/química , Listeria/inmunología , Listeria/patogenicidad , Linfopenia/complicaciones , Ratones , Ratones Endogámicos C57BLRESUMEN
Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression.