RESUMO
Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.
Assuntos
Técnicas de Cultura de Células/instrumentação , Meios de Cultura Livres de Soro/farmacologia , Células Matadoras Induzidas por Citocinas/citologia , Gases/química , Morte Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células Matadoras Induzidas por Citocinas/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Memória Imunológica/efeitos dos fármacos , Permeabilidade , FenótipoRESUMO
Introduction: Immunotherapy is now a standard treatment for many malignancies. Although immune checkpoint inhibition has demonstrated substantial efficacy by enhancing T cell activation and function in the tumor microenvironment, adoptive transfer of T and NK cell products promises to provide activated cells capable of immediate and direct tumor destruction. A widely applicable, non-MHC dependent, cellular therapy, consisting of in vitro generated dendritic cells (DC) combined with cytokine-induced killer cells (CIK), is highly efficient to produce from individual patients and has demonstrated safety and efficacy alone or with chemotherapy.Areas covered: We summarize the clinical data from studies of DC-CIK and discuss future research directions.Expert opinion: Patients with a wide variety of tumor types who have received DC-CIK therapy may experience clinical responses. This versatile therapy synergizes with other anti-cancer therapies including chemotherapy and immunotherapy.
Assuntos
Células Matadoras Induzidas por Citocinas/imunologia , Células Dendríticas/imunologia , Neoplasias/terapia , Terapia Combinada , Células Matadoras Induzidas por Citocinas/citologia , Células Dendríticas/citologia , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Taxa de Sobrevida , Microambiente TumoralRESUMO
We describe a method of rendering polyclonal cytokine-induced killer cells (CIK) specific against cytomegalovirus (CMV), focusing on GMP compliance. Peripheral blood mononuclear cells (PBMNC) are stimulated with pooled CMV peptides pp65 and IE-1 for 16-24â¯h and the reactive T cell subset which up-regulate CD137 is further co-stimulated with anti-CD137, followed by expansion in G-Rex flasks under standard CIK culture condition. This method generates a large number CMV-specific CIK with superior potency compared to published method currently in clinical trials. The cytotoxicity as measured by chromium release assay correlates with the upregulation of CD107a upon peptide re-challenge as measured by flow cytometry. CMV-CIK at maturity consist of mainly late effector memory CD8 T cells and have a skewed TCR repertoire with preferential expansion of a few families. Such CMV-CIK retain their function after freezing and thawing. CMV-CIK thus generated is ready for clinical trial against drug-resistant CMV disease.
Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/imunologia , Proteínas Imediatamente Precoces/imunologia , Memória Imunológica/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos T/citologia , Linfócitos T Citotóxicos/citologiaRESUMO
Epithelial ovarian cancer (EOC) is the most lethal of all gynecologic tumors. Cancer spheroid culture is a widely used model to study cancer stem cells. Previous studies have demonstrated the effectiveness of cytokineinduced killer (CIK) cellbased therapies against cancer and cancer stem cells. However, it is not clear how EOC spheroid cells respond to CIKmediated cellular lysis, and the mechanisms involved have never been reported before. A flow cytometrybased method was used to evaluate the anticancer effects of CIK cells against adherent A2780 cells and A2780 spheroids. To demonstrate the association between hypoxia inducible factor1α (HIF1A) and intercellular adhesion molecule1 (ICAM1), two HIF1A short hairpin RNA (shRNA) stable transfected cell lines were established. Furthermore, the protein expression levels of hypoxia/HIF1Aassociated signaling pathways were evaluated, including transforming growth factorß1 (TGFß1)/mothers against decapentaplegic homologs (SMADs) and nuclear factorκB (NFκB) signaling pathways, comparing A2780 adherent cells and cancer spheroids. Flow cytometry revealed that A2780 spheroid cells were more resistant to CIKmediated cellular lysis, which was partially reversed by an antiICAM1 antibody. HIF1A was significantly upregulated in A2780 spheroids compared with adherent cells. Using HIF1A shRNA stable transfected cell lines and cobalt chloride, it was revealed that hypoxia/HIF1A contributed to downregulation of ICAM1 in A2780 spheroid cells and adherent cells. Furthermore, hypoxia/HIF1Aassociated signaling pathways, TGFß1/SMADs and NFκB, were activated in A2780 spheroid cells by using western blotting. The findings indicate that EOC stemlike cells resist the CIKmediated cellular lysis via HIF1Amediated downregulation of ICAM1, which may be instructive for optimizing and enhancing CIKbased therapies.
Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Células Matadoras Induzidas por Citocinas/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Células-Tronco Neoplásicas/citologia , Neoplasias Ovarianas/metabolismo , Adulto , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Proliferação de Células , Células Matadoras Induzidas por Citocinas/transplante , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Molécula 1 de Adesão Intercelular/genética , Masculino , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapiaRESUMO
OBJECTIVE: Ex vivo expansion is an effective way to produce cytokine-induced killer (CIK) cells needed for clinical trials. Here, ex vivo expansion and metabolism characters of CIK cells in static and dynamic cultures and the relationship between cell expansion and metabolism were investigated. MATERIALS AND METHODS: Oxygen transfer efficiency was assessed by computational fluid dynamics technique. Cell phenotype, apoptosis and of transporter expression were determined by flow cytometry and Western blotting. Metabolites and enzyme activities were assessed by biochemical methods. RESULTS: Dynamic cultures favoured better CIK cell expansion without impairing their phenotype and cytotoxicity, enhanced oxygen transfer efficiency. The glucose metabolism flux of cells in dynamic cultures was enhanced by upregulating surface glucose transporter 1 expression and phosphofructokinase activity. Moreover, pentose phosphate pathway (PPP) metabolic flux was enhanced through upregulating glucose-6-phosphate dehydrogenase activity. Glutaminolysis was also accelerated via boosting glutamine transporters expression, glutaminase (GLS) and glutamate dehydrogenase activities. Together with higher oxygen consumption rate and extracellular acidification rate, it was suggested that cells in dynamic cultures were in a more vigorous metabolic state for ATP production. CONCLUSION: Dynamic cultures accelerated glucose and glutamine metabolic flux to promote ATP production, elevated glucose metabolic flux through PPP to promote biosynthesis for better cell expansion. These findings may provide the basis for ex vivo CIK cell expansion process optimization.
Assuntos
Trifosfato de Adenosina/biossíntese , Células Matadoras Induzidas por Citocinas/metabolismo , Via de Pentose Fosfato , Técnicas de Cultura de Células , Proliferação de Células , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/imunologia , Sangue Fetal/citologia , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glutamina/metabolismo , Glicólise , Humanos , Imunoterapia Adotiva , Modelos Biológicos , Neoplasias/imunologia , Neoplasias/terapia , Consumo de OxigênioRESUMO
BACKGROUND: Application of dendritic cells (DC) for cancer immunotherapy involves tumor-associated immunogenic antigens for effective therapeutic strategies. The present study investigated whether DC co-cultured with autologous cytokine-induced killer cells (CIK) could induce a more specific immune response against liver cancer stem cells (LCSC) generated from human hepatocellular carcinoma (HCC) cells in vitro and in vivo. METHODS: Human DC and CIK were generated from peripheral blood mononuclear cells (PBMCs) taken from consenting liver cancer patients. Flow cytometry was used to determine the phenotypes of DC and CIK, and cell proliferation. The tumor growth and anti-tumor activity of these cells were further evaluated using a nude mouse tumor model. RESULTS: We demonstrated that DC and CIK significantly enhanced the apoptosis ratio, depending on DC-CIK cell numbers, by increasing caspase-3 protein expression and reducing proliferating cell nuclear antigen (PCNA) protein expression against LCSC. The in vivo data indicated that DC-CIK exhibited significant LCSC cell-induced tumor growth inhibition in nude mice, which was most significant with LCSC antigen loaded DCs. CONCLUSIONS: The results showed, that DC-CIK cells could inhibit HCC and LCSC growths in vitro and in vivo and the most successful DC triggering of cell cytotoxic activity could be achieved by their LCSC antigen loading.
Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Células Matadoras Induzidas por Citocinas/imunologia , Células Dendríticas/imunologia , Imunoterapia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Adolescente , Adulto , Idoso , Animais , Antígenos de Neoplasias/imunologia , Carcinoma Hepatocelular/patologia , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Matadoras Induzidas por Citocinas/citologia , Células Dendríticas/citologia , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Adulto JovemRESUMO
Ex vivo expansion is an effective strategy to acquire cytokine-induced killer (CIK) cells needed for clinical trials. In this work, the effects of dynamic suspension culture, which was carried out by shake flasks on a shaker, on CIK cells were investigated by the analysis of expansion characteristics and physiological functions, with the objective to optimize the culture conditions for ex vivo expansion of CIK cells. The results showed that the expansion folds of total cells in dynamic cultures reached 69.36 ± 30.36 folds on day 14, which were significantly higher than those in static cultures (9.24 ± 1.12 folds, P < 0.05), however, the proportions of CD3+ cells and CD3+CD56+ cells in both cultures were similar, leading to much higher expansion of CD3+ cells and CD3+CD56+ cells in dynamic cultures. Additionally, expanded CIK cells in two cultures possessed comparable physiological functions. Notably, significantly higher percentages of CD25+ cells and CD69+ cells were found in dynamic cultures (P < 0.05). Besides, much higher glucose consumption rate of cells (P < 0.05) but similar YLac/gluc were observed in dynamic cultures. Further, cells in dynamic cultures had better glucose utilization efficiency. Together, these results suggested that dynamic cultures improved cell activation, then accelerated glucose consumption rate, which enhanced cell expansion and promoted glucose utilization efficiency of cells.
Assuntos
Técnicas de Cultura de Células/métodos , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/metabolismo , Glucose/metabolismo , Antígenos CD/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Células Matadoras Induzidas por Citocinas/fisiologia , Citometria de Fluxo , Humanos , Fatores de TempoRESUMO
Several clinical immunotherapy trials with cytokine-induced killer (CIK) cells have been reported. However, molecular evidence of cell expansion, acquisition of tumor cytotoxicity, and safety of CIK cells is required before putting them to clinical use. Here, we performed dynamic transcriptomic analyses of CIKs generated from primary peripheral blood mononuclear cells exposed to interferon-γ, OKT3, and interleukin-2. CIK mRNAs were extracted and sequenced at days 0, 1, 7, and 14 and subjected to bioinformatics analyses. Using weighted correlation network analysis (WGCNA), we identified two major gene modules that mediate immune cell activation and mitosis. We found that activation and cytotoxicity of CIK cells likely rely on cluster of differentiation 8 (CD8) and its protein partner LCK proto-oncogene, Src family tyrosine kinase (LCK). A time-course series analysis revealed that CIK cells have relatively low immunogenicity because of decreased expression of some self-antigens. Importantly, we identified several crucial activating receptors and auxiliary adhesion receptors expressed on CIK cells that may function as tumor sensors. Interestingly, cytotoxicity-associated genes, including those encoding PRF1, GZMB, FASL, and several cytokines, were up-regulated in mature CIK cells. Most immune-checkpoint molecules and inflammatory tumor-promoting factors were down-regulated in the CIK cells, suggesting efficacy and safety in future clinical trials. Notably, insulin-like growth factor 1 (IGF-1) was highly expressed in CIK cells and may promote cytotoxicity, although it also could facilitate tumorigenesis. The transcriptomic atlas of CIK cells presented here may inform efforts to improve CIK-associated tumor cytotoxicity and safety in clinical trials.
Assuntos
Células Matadoras Induzidas por Citocinas/metabolismo , Perfilação da Expressão Gênica , Ciclo Celular/genética , Ciclo Celular/imunologia , Linhagem Celular , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/imunologia , Humanos , Imunoterapia/efeitos adversos , Família Multigênica/genética , Família Multigênica/imunologia , Proto-Oncogene Mas , Segurança , Análise de Sequência de RNARESUMO
BACKGROUND: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19+ malignancies. METHODS: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs. RESULTS: CB-CIKs, like PB-CIKs, were mostly CD3+ T cells with mean 45% CD3+CD56+ and expressing mostly TCR(T cell receptor)αß with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4+ cells, mostly CD56-, as well as a greater proportion of naïve CCR7+CD45RA+ and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8+ cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19+ tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph+ CD19+ acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8â¯×â¯106 CIK/kg. DISCUSSION: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/transplante , Sangue Fetal/citologia , Neoplasias/terapia , Animais , Antígenos CD19/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Terapia Combinada , Células Matadoras Induzidas por Citocinas/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/fisiologia , Feminino , Sangue Fetal/imunologia , Humanos , Imunoterapia Adotiva/métodos , Recém-Nascido , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/transplante , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Resultado do TratamentoRESUMO
Ovarian cancer stem cells (OCSCs) are highly carcinogenic and have very strong resistance to traditional chemotherapeutic drugs; therefore, they are an important factor in ovarian cancer metastasis and recurrence. It has been reported that dendritic cell (DC)-cytokine-induced killer (CIK) cells have significant killing effects on all cancer cells across many systems including the blood, digestive, respiratory, urinary and reproductive systems. However, whether DC-CIK cells can selectively kill OCSCs is currently unclear. In this study, we collected ovarian cancer patient menstrual blood (OCPMB) samples to acquire mononuclear cells and isolated DC-CIK cells in vitro. In addition, autologous CD44+/CD133+ OCSCs were isolated and used as target cells. The experimental results showed that when DC-CIK cells and OCSCs were mixed and cultured in vitro at ratios of 5:1, 10:1 and 50:1, the DC-CIK cells killed significant amounts of OCSCs, inhibited their invasion in vitro and promoted their apoptosis. The qPCR and Western blot results showed that DC-CIK cells stimulated high expression levels and phosphorylation of TNFR1, ASK1, AIP1 and JNK in OCSCs through the release of TNF-α. After the endogenous TNFR1 gene was knocked out in OCSCs using the CRISPR/Cas9 technology, the killing function of DC-CIK cells on target OCSCs was significantly attenuated. The results of the analyses of clinical samples suggested that the TNFR1 expression level was negatively correlated with ovarian cancer stage and prognosis. Therefore, we innovatively confirmed that DC-CIK cells derived from OCPMB could secret TNF-α to activate the expression of the TNFR1-ASK1-AIP1-JNK pathway in OCSCs and kill autologous OCSCs.