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Introduction: Intra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells. Methods: B lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry. Results: Antibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients' sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells. Discussion: These studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.
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Ascitis , Linfocitos B , Herpesvirus Humano 4 , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/inmunología , Herpesvirus Humano 4/inmunología , Linfocitos B/inmunología , Ascitis/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Latencia del Virus/inmunología , Microambiente Tumoral/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Carcinoma Epitelial de Ovario/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular TumoralRESUMEN
Cell migration regulated by Thrombospondin 2 (THSB2) is important for the development of pulmonary artery remodeling, but the mechanism by which THBS2-mediated cell migration regulates the development of pulmonary artery remodeling in broiler ascites syndrome (AS) is unclear. In addition, the lack of chicken THBS2 antibodies makes it difficult to study the mechanism in depth. In our study, we used recombinant gene technology, protein purification, and other techniques to obtain mouse anti-chicken THBS2 antibody and analyze its expression in broilers, ascites broilers and other animals. The results showed that we immunized mouse with recombinant THBS2 protein and obtained an antibody titer of 1:204,800, and the addition of astragalus polysaccharide as an immunomodulator during immunization significantly increased the titer of the antibody. Western blotting (WB) and immunofluorescence results showed that the THBS2 was significantly down-regulated in the ascites broiler. The THBS2 antibody we prepared can also detect THBS2 protein in duck, mouse, goat, and rabbit tissues. These results provide a foundation for further investigation of the role of THBS2 in pulmonary artery remodeling in broiler ascites syndrome and a powerful tool for studying the role of THBS2 in AS.
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Anticuerpos , Pollos , Hipertensión Pulmonar , Proteínas Recombinantes , Trombospondinas , Animales , Proteínas Recombinantes/inmunología , Trombospondinas/inmunología , Trombospondinas/genética , Ratones , Hipertensión Pulmonar/inmunología , Anticuerpos/inmunología , Ascitis/inmunología , Arteria Pulmonar , Enfermedades de las Aves de Corral/inmunologíaRESUMEN
Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.
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Apirasa , Linfocitos T CD8-positivos , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Apirasa/metabolismo , Apirasa/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Persona de Mediana Edad , Ascitis/inmunología , Ascitis/patología , Ascitis/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Anciano , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/antagonistas & inhibidores , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Antígenos HLA-DR/metabolismo , Adulto , Agotamiento de Células T , Proteínas del Grupo de Alta MovilidadRESUMEN
BACKGROUNDS: The Gustave Roussy Immune (GRIm) score predicts survival outcomes in several cancers. However, the prognostic significance of the GRIm score in patients with malignant ascites has not yet been investigated. METHODS: Clinical samples were collected from a cohort of patients with malignant ascites secondary to hepatocellular carcinoma (HCC). We calculated serum GRIm (sGRIm) and ascites GRIm (aGRIm) scores and divided the samples into low and high GRIm score groups. Survival analysis was used to compare the prognostic significance of the sGRIm and aGRIm scores. 16S rRNA sequencing was performed to determine the profiles of the intratumoral microbiota in the groups. A fluorescent multiplex immunohistochemistry (mIHC) assay was used to detect the expression of different immune cells by labeling seven markers of malignant ascites. RESULTS: 155 patients with HCC and malignant ascites were enrolled in this study. Survival analysis revealed that the aGRIm score showed a superior prognostic significance compared to the sGRIm score. Microbial analysis demonstrated that the bacterial richness and diversity were higher in the low aGRIm score group than in the high aGRIm score group. LefSe analysis revealed that certain bacteria were correlated with high aGRIm scores. Fluorescent mIHC displayed the tumor microenvironment of malignant ascites and found that the density of CD8 + T cells was significantly higher in the low aGRIm score group than in the high aGRIm score group. CONCLUSIONS: Our present study identified a novel scoring system (aGRIm score) that can predict the survival outcome of patients with malignant ascites secondary to HCC.
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Ascitis , Carcinoma Hepatocelular , Neoplasias Hepáticas , Microbiota , Humanos , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/mortalidad , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Ascitis/inmunología , Ascitis/microbiología , Femenino , Masculino , Persona de Mediana Edad , Microbiota/inmunología , Anciano , Pronóstico , Microambiente Tumoral/inmunología , Adulto , ARN Ribosómico 16S/genéticaRESUMEN
Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.
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Citotoxicidad Celular Dependiente de Anticuerpos , Ascitis , Células Asesinas Naturales , Neoplasias Ováricas , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ascitis/inmunología , Femenino , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Línea Celular Tumoral , Inmunoglobulinas/metabolismo , Receptores de IgG/metabolismo , Receptores de IgG/inmunología , Degranulación de la Célula/inmunología , Degranulación de la Célula/efectos de los fármacos , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Cetuximab/farmacologíaRESUMEN
Objective: To observe the level and detection of ascites CD100 on the activity of CD4(+) and CD8(+) T lymphocytes in vitro in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis. Methods: Peripheral blood and ascites were collected from 77 cases of liver cirrhosis (49 patients with liver cirrhosis combined with simple ascites and 28 patients with liver cirrhosis combined with SBP), and peripheral blood was collected from 22 controls. Soluble CD100 (sCD100) in peripheral blood and ascites was detected by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect membrane-bound CD100 (mCD100) on the surface of CD4(+) and CD8(+)T lymphocytes. CD4(+) and CD8(+)T lymphocytes in ascites were sorted. CD4(+)T lymphocyte proliferation, key transcription factor mRNA, and secreted cytokine changes, as well as CD8(+)T lymphocyte proliferation, important toxic molecule mRNA, and secreted cytokine changes, were detected after CD100 stimulation. The killing activity of CD8(+)T cells was detected by direct contact and indirect contact culture systems. Data conforming to normality were compared using one-way ANOVA, a student's t-test, or a paired t-test. Data that did not conform to a normal distribution were compared using either the Krusal-Willis test or the Mann-Whitney test. Results: There was no statistically significant difference in plasma sCD100 level between patients with liver cirrhosis combined simple ascites (1 415 ± 434.1) pg/ml, patients with liver cirrhosis combined with SBP (1 465 ± 386.8) pg/ml, and controls (1 355 ± 428.0) pg/ml (P = 0.655). The ascites sCD100 level was lower in patients with liver cirrhosis combined with SBP than that of patients with simple ascites [(2 409 ± 743.0) pg/ml vs. (2825±664.2) pg/ml, P=0.014]. There was no statistically significant difference in the level of mCD100 in peripheral blood CD4(+) and CD8(+) T lymphocytes among the three groups (P > 0.05). The levels of mCD100 in ascites CD4(+) and CD8(+) T lymphocytes were higher in patients with liver cirrhosis combined with SBP than those in patients with simple ascites (P < 0.05). CD100 stimulation had no significant effect on the proliferation of CD4(+) and CD8(+)T lymphocytes in the ascites of patients with liver cirrhosis combined with SBP (P > 0.05). There were no significant effects on the expression of transcription factors in effector CD4(+)T lymphocytes (T-bet, retinoic acid associated solitary nuclear receptor γt, aromatic hydrocarbon receptor) or secretion of cytokines (interferon-γ, 17, and 22) (P > 0.05). CD100 stimulation had increased the relative expression of perforin, granzyme B, and granlysin mRNA and the levels of secreted interferon-γ and tumor necrosis factor-α, killing activity in ascites CD8+ T lymphocytes of patients with liver cirrhosis combined with SBP (P < 0.05). Conclusion: The active form of CD100 is sCD100 instead of mCD100. There is an imbalance between the expression of sCD100 and mCD100 in the ascites of patients with cirrhosis combined with SBP. sCD100 can enhance the function of CD8(+)T lymphocytes in the ascites of patients with cirrhosis combined with SBP and thus is one of the potential therapeutic targets.
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Antígenos CD , Ascitis , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Cirrosis Hepática , Peritonitis , Ascitis/inmunología , Inmunomodulación/inmunología , Antígenos CD/sangre , Antígenos CD/inmunología , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Cirrosis Hepática/inmunología , Peritonitis/sangre , Peritonitis/complicaciones , Peritonitis/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , HumanosRESUMEN
BACKGROUND: Ovarian cancer (OC) is typically diagnosed late, associated with high rates of metastasis and the onset of ascites during late stage disease. Understanding the tumor microenvironment and how it impacts the efficacy of current treatments, including immunotherapies, needs effective in vivo models that are fully characterized. In particular, understanding the role of immune cells within the tumor and ascitic fluid could provide important insights into why OC fails to respond to immunotherapies. In this work, we comprehensively described the immune cell infiltrates in tumor nodules and the ascitic fluid within an optimized preclinical model of advanced ovarian cancer. METHODS: Green Fluorescent Protein (GFP)-ID8 OC cells were injected intraperitoneally into C57BL/6 mice and the development of advanced stage OC monitored. Nine weeks after tumor injection, mice were sacrificed and tumor nodules analyzed to identify specific immune infiltrates by immunohistochemistry. Ascites, developed in tumor bearing mice over a 10-week period, was characterized by mass cytometry (CyTOF) to qualitatively and quantitatively assess the distribution of the immune cell subsets, and their relationship to ascites from ovarian cancer patients. RESULTS: Tumor nodules in the peritoneal cavity proved to be enriched in T cells, antigen presenting cells and macrophages, demonstrating an active immune environment and cell-mediated immunity. Assessment of the immune landscape in the ascites showed the predominance of CD8+ , CD4+ , B- , and memory T cells, among others, and the coexistance of different immune cell types within the same tumor microenvironment. CONCLUSIONS: We performed, for the first time, a multiparametric analysis of the ascitic fluid and specifically identify immune cell populations in the peritoneal cavity of mice with advanced OC. Data obtained highlights the impact of CytOF as a diagnostic tool for this malignancy, with the opportunity to concomitantly identify novel targets, and define personalized therapeutic options.
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Neoplasias Ováricas/inmunología , Microambiente Tumoral/inmunología , Animales , Ascitis/inmunología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Alcohol-associated hepatitis (AAH) is a severe form of liver injury with mortality as high as 30%-40% at 90 days. As a result of altered immune function in AAH, bacterial infections are common and are associated with poor outcomes. However, determining the risk and subsequent development of infection in patients with AAH remain challenging. We performed a retrospective study of consecutive patients admitted with a diagnosis of AAH at two independent tertiary centers from 1998 to 2018 (test cohort, n = 286) who developed infections following hospitalization. The diagnosis of AAH was confirmed by manual chart review according to the recent National Institute on Alcohol Abuse and Alcoholism definition. Infections were categorized by location and time of diagnosis as hospital-acquired infection (48 hours after admission until discharge) and posthospital infections (up to 6 months following discharge). The cohort was 66% men, and the median age was 48 (21-83) years. Corticosteroids were used in 32% of all patients with AAH. The overall infection rate was 24%. Of those with infections, 46% were hospital acquired and 54% were acquired after hospitalization. Variables found to be significant risk factors for bacterial infection included the presence of ascites on admission (hazard ratio [HR], 2.06), corticosteroid administration (HR, 1.70), Model for End-Stage Liver Disease (MELD) >23 (HR, 2.61), and white blood cell (WBC) count on admission per point (HR, 1.02). Conclusion: In this multicenter cohort study of patients hospitalized with AAH, MELD score, ascites, WBC count, and use of corticosteroids were identified as significant predictors of the development of bacterial infection. We created a novel predictive equation that may be used to aid in the identification of patients with AAH at high risk of infection.
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Infecciones Bacterianas/epidemiología , Infección Hospitalaria/epidemiología , Hepatitis Alcohólica/microbiología , Alta del Paciente/estadística & datos numéricos , Medición de Riesgo , Corticoesteroides/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Ascitis/inmunología , Ascitis/microbiología , Infecciones Bacterianas/inmunología , Infección Hospitalaria/inmunología , Femenino , Hepatitis Alcohólica/inmunología , Hospitalización/estadística & datos numéricos , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Índice de Severidad de la Enfermedad , Centros de Atención Terciaria , Adulto JovenRESUMEN
Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.
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Ascitis/inmunología , Antígeno CD56/inmunología , Cistadenocarcinoma Seroso/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Apirasa/genética , Apirasa/inmunología , Ascitis/genética , Ascitis/patología , Antígeno CD56/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunofenotipificación , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-15/genética , Interleucina-15/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Células K562 , Células Asesinas Naturales/patología , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/patología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
BACKGROUND: Gastric cancer (GC) patients with peritoneal metastasis usually have extremely poor prognosis. Intraperitoneal infusion of paclitaxel (PTX) provides an effective treatment, but relapse and PTX-resistance are unavoidable disadvantages, and it is difficult to monitor the occurrence of PTX-resistance. OBJECTIVE: The aim of this study was to explore novel autoantibodies in the ascites of individuals with relapsed PTX-resistant GC with peritoneal metastasis. METHODS: Ascites samples were collected before PTX infusion and after the relapse in 3 GC patients. To determine the expression of significantly changed proteins, we performed autoantibody profiling with immunome protein microarrays and tandem mass tag (TMT) quantitative proteomics, and then, the overlapping proteins were selected. RESULTS: Thirty-eight autoantibodies that were differentially expressed between the ascites in the untreated group and relapsed PTX-resistant group were identified. For confirmation of the results, TMT quantitative proteomics was performed, and 842 dysregulated proteins were identified. Four proteins, TPM3, EFHD2, KRT19 and vimentin, overlapped between these two assays. CONCLUSIONS: Our results first revealed that TPM3, EFHD2, KRT19 and vimentin were novel autoantibodies in the ascites of relapsed PTX-resistant GC patients. These autoantibodies may be used as potential biomarkers to monitor the occurrence of PTX-resistance.
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Ascitis/inmunología , Autoanticuerpos/análisis , Neoplasias Peritoneales/secundario , Neoplasias Gástricas/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Autoanticuerpos/inmunología , Resistencia a Antineoplásicos , Humanos , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/patología , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patologíaRESUMEN
Chemorefractory ovarian cancer has limited therapeutic options. Hence, new types of treatment including neoantigen-specific immunotherapy need to be investigated. Neoantigens represent promising targets for personalized cancer immunotherapy. We here describe the clinical and immunological effects of a neoantigen peptide-loaded DC-based immunotherapy in a patient with recurrent and chemoresistant ovarian cancer. A 71-year-old female patient with chemorefractory ovarian cancer and malignant ascites received intranodal vaccination of DCs loaded with four neoantigen peptides that were predicted by our immunogenomic pipeline. Following four rounds of vaccinations with this therapy, CA-125 levels were remarkably declined and tumor cells in the ascites were also decreased. Concordantly, the tumor-related symptoms such as respiratory discomfort improved without any adverse reactions. The reactivity against one HLA-A2402-restricted neoantigen peptide derived from a mutated PPM1 F protein was detected in lymphocytes from peripheral blood by IFN-γ ELISPOT assay. Furthermore, the neoantigen (PPM1 F mutant)-specific TCRs were detected in the tumor-infiltrating T lymphocytes post-vaccination. Our results showed that vaccination with intranodal injection of neoantigen peptide-loaded DCs may have clinical and immunological impacts on cancer treatment.
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Ascitis/terapia , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Ováricas/terapia , Ganglio Linfático Centinela/inmunología , Linfocitos T/inmunología , Anciano , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Ascitis/inmunología , Antígeno Ca-125/sangre , Resistencia a Antineoplásicos , Ensayo de Immunospot Ligado a Enzimas , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Neoplasias Ováricas/inmunología , Péptidos/inmunología , Carga Tumoral , VacunaciónAsunto(s)
Ascitis/diagnóstico , Carcinoma Papilar/diagnóstico , Enfermedad Relacionada con Inmunoglobulina G4/diagnóstico , Neoplasias Quísticas, Mucinosas y Serosas/diagnóstico , Neoplasias Peritoneales/diagnóstico , Anciano , Ascitis/inmunología , Ascitis/terapia , Biomarcadores/sangre , Diagnóstico Diferencial , Femenino , Humanos , Inmunoglobulina G/sangre , Enfermedad Relacionada con Inmunoglobulina G4/inmunología , Enfermedad Relacionada con Inmunoglobulina G4/terapia , Valor Predictivo de las PruebasRESUMEN
Despite the role of curcumin in controlling inflammation, angiogenesis, and cancer in human cells, its therapeutic use is limited. The reasons are quick metabolic breakdown, low aqueous solubility, and bioavailability. This study describes the advantages of clinical-grade curcumin-incorporated fibrin matrix either in lyophilized off-the-shelf wafer or injectable hydrogel forms, as a biodegradable local delivery system. To produce the curcumin-fibrin wafer, used clinical-grade fibrin sealant in a modified composition. To fabricate wafer, we premixed the curcumin with either fibrinogen or thrombin, before clotting into a hydrogel. Sustained release of active curcumin from fibrin wafer, suspended in culture medium at 37 °C lasted for seven days. Upon premixing albumin with thrombin and subsequently adding curcumin into the mixture improved the loading concentration and stability. Dose- and time-dependent apoptotic function of curcumin on cancer cell lines upon release from fibrin wafer, were demonstrated in vitro. In vivo immuno-modulation and a nontoxic response to curcumin released from fibrin into the peritoneal cavity of mice were established. The cytotoxic effect of released curcumin was demonstrated; showing both a preventive and therapeutic role against tumor growth. In vivo studies used Dalton's Lymphoma Ascites (DLA) mice model. Both implanted fibrin wafer and injected hydrogel can breakdown by a physiological process and get cleared by the fibrinolytic mechanism. The lyophilized fibrin wafer could function as a hemostat, adhere to surgical cancer tissues, and arrest bleeding. The potential of curcumin in preventing solid tumor metastasis may be explored upon the sustained delivery of the molecule from the fibrin wafer.
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Antineoplásicos Fitogénicos/farmacología , Ascitis/tratamiento farmacológico , Curcumina/farmacología , Portadores de Fármacos , Fibrina/química , Linfoma/tratamiento farmacológico , Células A549 , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Ascitis/inmunología , Ascitis/patología , Proliferación Celular/efectos de los fármacos , Curcumina/química , Preparaciones de Acción Retardada , Composición de Medicamentos , Liberación de Fármacos , Femenino , Humanos , Hidrogeles , Linfoma/inmunología , Linfoma/patología , Ratones , Células PC-3RESUMEN
Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic ß-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness.
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Carcinoma Epitelial de Ovario/inmunología , Macrófagos Peritoneales/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Peritoneales/inmunología , Escape del Tumor/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Anciano , Animales , Ascitis/genética , Ascitis/inmunología , Ascitis/patología , Carcinoma Epitelial de Ovario/mortalidad , Carcinoma Epitelial de Ovario/secundario , Carcinoma Epitelial de Ovario/terapia , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia Adoptiva/métodos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Persona de Mediana Edad , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Comunicación Paracrina/inmunología , Neoplasias Peritoneales/mortalidad , Neoplasias Peritoneales/secundario , Cultivo Primario de Células , Pronóstico , Receptores Quiméricos de Antígenos/inmunología , Esferoides Celulares/inmunología , Esferoides Celulares/metabolismo , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rß-selective IL2/anti-IL2 complexes (IL2c) to stimulate effector T cells preferentially in the orthotopic mouse ID8agg ovarian cancer model. Despite strong tumor rejection, IL2c unexpectedly lowered the tumor microenvironmental CD8+/Treg ratio. IL2c reduced tumor microenvironmental Treg suppression and induced a fragile Treg phenotype, helping explain improved efficacy despite numerically increased Tregs without affecting Treg in draining lymph nodes. IL2c also reduced Treg-mediated, high-affinity IL2R signaling needed for optimal Treg functions, a likely mechanism for reduced Treg suppression. Effector T-cell IL2R signaling was simultaneously improved, suggesting that IL2c inhibits Treg functions without hindering effector T cells, a limitation of most Treg depletion agents. Anti-PD-L1 antibody did not treat ID8agg, but adding IL2c generated complete tumor regressions and protective immune memory not achieved by either monotherapy. Similar anti-PD-L1 augmentation of IL2c and degradation of Treg functions were seen in subcutaneous B16 melanoma. Thus, IL2c is a multifunctional immunotherapy agent that stimulates immunity, reduces immunosuppression in a site-specific manner, and combines with other immunotherapies to treat distinct tumors in distinct anatomic compartments. SIGNIFICANCE: These findings present CD122-targeted IL2 complexes as an advancement in cancer immunotherapy, as they reduce Treg immunosuppression, improve anticancer immunity, and boost PD-L1 immune checkpoint blockade efficacy in distinct tumors and anatomic locations.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Inmunoterapia/métodos , Subunidad beta del Receptor de Interleucina-2/antagonistas & inhibidores , Interleucina-2/farmacología , Melanoma Experimental/terapia , Neoplasias Ováricas/terapia , Linfocitos T Reguladores/citología , Animales , Ascitis/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Femenino , Tolerancia Inmunológica , Inmunidad Celular , Memoria Inmunológica , Subunidad beta del Receptor de Interleucina-2/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/inmunología , Fenotipo , Distribución Aleatoria , Receptores de Interleucina-2/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunologíaAsunto(s)
Ascitis/diagnóstico , Enteritis/diagnóstico , Eosinofilia/diagnóstico , Gastritis/diagnóstico , Derrame Pericárdico/diagnóstico , Derrame Pleural/diagnóstico , Adulto , Ascitis/sangre , Ascitis/tratamiento farmacológico , Ascitis/inmunología , Duodeno/diagnóstico por imagen , Duodeno/inmunología , Duodeno/patología , Ecocardiografía , Endoscopía Gastrointestinal , Enteritis/complicaciones , Enteritis/tratamiento farmacológico , Enteritis/inmunología , Eosinofilia/complicaciones , Eosinofilia/tratamiento farmacológico , Eosinofilia/inmunología , Eosinófilos/inmunología , Femenino , Gastritis/complicaciones , Gastritis/tratamiento farmacológico , Gastritis/inmunología , Glucocorticoides/uso terapéutico , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Recuento de Leucocitos , Derrame Pericárdico/sangre , Derrame Pericárdico/tratamiento farmacológico , Derrame Pericárdico/inmunología , Derrame Pleural/sangre , Derrame Pleural/tratamiento farmacológico , Derrame Pleural/inmunología , Recto/diagnóstico por imagen , Recto/inmunología , Recto/patología , Resultado del TratamientoRESUMEN
Efficacy for alleviating signs/symptoms of malignant ascites of a renovated CART (cell-free and concentrated ascites reinfusion therapy) system, called KM-CART, was evaluated. A total of 4781 KM-CART procedures were performed in 2109 patients. All patients were accepted unless hemodynamically unstable or consciousness impaired. The ascites were processed and drip-infused into the patient. There were no major complications or deaths. The mean drainage volume was 6.2 L (maximum: 27.7 L), patient symptoms (numerical scale system) were significantly alleviated (45.1 ± 19.0 reduced to 21.2 ± 14.2, P < .001), and patient leg circumference significantly decreased (33.3 ± 4.4 cm reduced to 30.5 ± 4.4 cm, P < .001) without exacerbation of renal function. Collected cancer cells could be utilized for immune therapy. KM-CART is capable of improving the "quality of best supportive care" and can be beneficial in conjunction with medication for alleviating malignant pain.
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Ascitis/terapia , Líquido Ascítico/inmunología , Drenaje/métodos , Infusiones Parenterales/métodos , Neoplasias/terapia , Adulto , Anciano , Anciano de 80 o más Años , Ascitis/inmunología , Terapia Combinada/efectos adversos , Terapia Combinada/métodos , Drenaje/efectos adversos , Estudios de Factibilidad , Femenino , Humanos , Infusiones Parenterales/efectos adversos , Masculino , Persona de Mediana Edad , Neoplasias/complicaciones , Neoplasias/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
Simultaneously targeted treatment of tumor cells and their surrounding growth-supporting immune cells is a promising strategy to reshape immunosuppressive tumor microenvironment (TME) and potentiate host innate and adaptive antitumor immune responses. Methods: We designed a series of melittin-(RADA)n hybrid peptide sequences with varying self-assembling motifs of RADA and screened out a melittin-(RADA)6 peptide that has an optimal gel-formation ability and in vitro antitumor activity. Results: The formed melittin-(RADA)6 (MR52) hydrogel scaffold could be loaded with a specific Ca2+/calmodulin-dependent protein kinase II (CAMKII) inhibitor, KN93, originally found to have both direct tumoricidal activity and macrophages-reprogramming ability, for potent immunotherapy against melanoma and hepatoma ascites in mice models. Our MR52 hydrogel has an interweaving nanofiber-like structure, possesses direct antitumor and controlled drug release properties, and promotes the enhanced intracellular uptake of loaded cargo. Compared to free KN93, the MR52-KN93 hydrogel (MRK) improved the killing effects and levels of immunogenic cell death (ICD) on tumor cells significantly. Due to the dual role of KN93, the injection of the MRK hydrogel retarded the growth of subcutaneous melanoma tumors dramatically and resulted in a high number of mature dendritic cells of draining lymph nodes, significantly enhancing the portion of cytotoxic T cells and reduced number of M2-like tumor-associated macrophages (TAMs) in tumors. Using a mouse model of malignant ascites (MAs), where traditional therapy was ineffective, we demonstrated that the MRK hydrogel treatment offered a significantly prolonged survival compared to controls. Following treatment with the MRK hydrogel, macrophages had elevated programmed cell death protein ligand-1 (PD-L1) expression, promising follow-up combined anti-PD-1 therapy that confers a cure rate of approximately 30% against MAs in mice models. Conclusion: Thus, the MRK hydrogel may serve as a prospective platform for antitumor applications.
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Antineoplásicos/uso terapéutico , Ascitis/terapia , Bencilaminas/uso terapéutico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Hidrogeles/administración & dosificación , Inmunoterapia/métodos , Neoplasias Hepáticas Experimentales/terapia , Melanoma Experimental/terapia , Meliteno/administración & dosificación , Terapia Molecular Dirigida/métodos , Proteínas de Neoplasias/antagonistas & inhibidores , Oligopéptidos/administración & dosificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Sulfonamidas/uso terapéutico , Macrófagos Asociados a Tumores/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antineoplásicos/administración & dosificación , Ascitis/etiología , Ascitis/inmunología , Antígeno B7-H1/biosíntesis , Bencilaminas/administración & dosificación , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Técnicas de Reprogramación Celular , Composición de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Inyecciones Intraperitoneales , Neoplasias Hepáticas Experimentales/complicaciones , Neoplasias Hepáticas Experimentales/inmunología , Activación de Macrófagos , Masculino , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas de Neoplasias/fisiología , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Proteínas Recombinantes de Fusión/administración & dosificación , Sulfonamidas/administración & dosificación , Escape del Tumor/efectos de los fármacos , Macrófagos Asociados a Tumores/clasificación , Macrófagos Asociados a Tumores/enzimologíaRESUMEN
BACKGROUND AND AIMS: Patients with advanced liver cirrhosis have an increased susceptibility to infections. As part of the cirrhosis-associated immune dysfunction, mucosal-associated invariant T (MAIT) cells, which have the capacity to respond to bacteria, are severely diminished in circulation and liver tissue. However, MAIT cell presence and function in the peritoneal cavity, a common anatomical site for infections in cirrhosis, remain elusive. In this study, we deliver a comprehensive investigation of the immune compartment present in ascites of patients with decompensated liver cirrhosis, and focus especially on MAIT cells. APPROACH AND RESULTS: To study this, matched peripheral blood and ascites fluid were collected from 35 patients with decompensated cirrhosis, with or without spontaneous bacterial peritonitis (SBP). MAIT cell phenotype and function were analyzed using high-dimensional flow cytometry, and the obtained data were compared with the blood samples of healthy controls (n = 24) and patients with compensated cirrhosis (n = 11). We found circulating MAIT cells to be severely decreased in patients with cirrhosis as compared with controls. In contrast, in ascites fluid, MAIT cells were significantly increased together with CD14+ CD16+ monocytes, innate lymphoid cells, and natural killer cells. This was paralleled by elevated levels of several pro-inflammatory cytokines and chemokines in ascites fluid as compared with plasma. Peritoneal MAIT cells displayed an activated tissue-resident phenotype, and this was corroborated by increased functional responses following stimulation with E. coli or interleukin (lL)-12 + IL-18 as compared with circulating MAIT cells. During SBP, peritoneal MAIT cell frequencies increased most among all major immune cell subsets, suggestive of active homing of MAIT cells to the site of infection. CONCLUSIONS: Despite severely diminished MAIT cell numbers and impaired phenotype in circulation, peritoneal MAIT cells remain abundant, activated, and highly functional in decompensated cirrhosis and are further enriched in SBP. This suggests that peritoneal MAIT cells could be of interest for immune-intervention strategies in patients with decompensated liver cirrhosis and SBP.
Asunto(s)
Ascitis/inmunología , Cirrosis Hepática/inmunología , Células T Invariantes Asociadas a Mucosa/fisiología , Adulto , Anciano , Infecciones Bacterianas/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peritonitis/inmunología , FenotipoRESUMEN
BACKGROUND: Rifaximin has been shown to reduce the incidence of hepatic encephalopathy and other complications in patients with cirrhosis. However, few studies have investigated the effect of rifaximin in cirrhotic patients with refractory ascites. AIM: To evaluate the effects of rifaximin in the treatment of refractory ascites and to preliminarily explore its possible mechanism. METHODS: A total of 75 cirrhotic patients with refractory ascites were enrolled in the study (50 in a rifaximin and 25 in a control group). Patients in the rifaximin group were divided into two subgroups according to the presence of spontaneous bacterial peritonitis and treatment with or without other antibiotics (19 patients treated with rifaximin and 31 patients treated with rifaximin plus intravenous antibiotics). All patients received conventional treatment for refractory ascites, while patients in the rifaximin group received oral rifaximin-α 200 mg four times daily for at least 2 wk. The ascites grade, fasting weight, liver and kidney function, and inflammatory factors in the plasma were evaluated before and after treatment. In addition, the gut microbiota was determined by metagenomics sequencing to analyse the changes in the characteristics of the gut microbiota before and after rifaximin treatment. The patients were followed for 6 mo. RESULTS: Compared with the control group, the fasting weight of patients significantly decreased and the ascites significantly subsided after treatment with rifaximin (P = 0.011 and 0.009, respectively). The 6-mo survival rate of patients in the rifaximin group was significantly higher than that in the control group (P = 0.048). The concentration of interferon-inducible protein 10 decreased significantly in the rifaximin group compared with that in the control group (P = 0.024). The abundance of Roseburia, Haemophilus, and Prevotella was significantly reduced after rifaximin treatment, while the abundance of Lachnospiraceae_noname, Subdoligranulum, and Dorea decreased and the abundance of Coprobacillus increased after treatment with rifaximin plus intravenous antibiotics. The gene expression of virulence factors was significantly reduced after treatment in both subgroups treated with rifaximin or rifaximin plus intravenous antibiotics. CONCLUSION: Rifaximin mitigates ascites and improves survival of cirrhotic patients with refractory ascites. A possible mechanism is that rifaximin regulates the structure and function of intestinal bacteria, thus improving the systemic inflammatory state.