CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

Preclinical assessment of CAR-NK cell-mediated killing efficacy and pharmacokinetics in a rapid zebrafish xenograft model of metastatic breast cancer.

Natural killer (NK) cells are attractive effectors for adoptive immunotherapy of cancer. Results from first-in-human studies using chimeric antigen receptor (CAR)-engineered primary NK cells and NK-92 cells are encouraging in terms of efficacy and safety. In order to further improve treatment strategies and to test the efficacy of CAR-NK cells in a personalized manner, preclinical screening assays using patient-derived tumor samples are needed. Zebrafish (Danio rerio) embryos and larvae represent an attractive xenograft model to study growth and dissemination of patient-derived tumor cells because of their superb live cell imaging properties. Injection into the organism's circulation allows investigation of metastasis, cancer cell-to-immune cell-interactions and studies of the tumor cell response to anti-cancer drugs. Here, we established a zebrafish larval xenograft model to test the efficacy of CAR-NK cells against metastatic breast cancer in vivo by injecting metastatic breast cancer cells followed by CAR-NK cell injection into the Duct of Cuvier (DoC). We validated the functionality of the system with two different CAR-NK cell lines specific for PD-L1 and ErbB2 (PD-L1.CAR NK-92 and ErbB2.CAR NK-92 cells) against the PD-L1-expressing MDA-MB-231 and ErbB2-expressing MDA-MB-453 breast cancer cell lines. Injected cancer cells were viable and populated peripheral regions of the larvae, including the caudal hematopoietic tissue (CHT), simulating homing of cancer cells to blood forming sites. CAR-NK cells injected 2.5 hours later migrated to the CHT and rapidly eliminated individual cancer cells throughout the organism. Unmodified NK-92 also demonstrated minor in vivo cytotoxicity. Confocal live-cell imaging demonstrated intravascular migration and real-time interaction of CAR-NK cells with MDA-MB-231 cells, explaining the rapid and effective in vivo cytotoxicity. Thus, our data suggest that zebrafish larvae can be used for rapid and cost-effective in vivo assessment of CAR-NK cell potency and to predict patient response to therapy.

Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.

Introduction: Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. Methods: In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. Results: We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Conclusion: Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Live-cell imaging for analysis of the NK cell immunological synapse.

The immunological synapse (IS) between NK cells and cancer cells is instrumental for the initiation of tumor-specific cytotoxicity. Improper function of processes at the IS can lead to NK cell unresponsiveness, contributing to tumor immune escape. Critical steps at the IS include target cell recognition, conjugation of NK cell and cancer cell, cytotoxic granule convergence to the microtubule-organizing center (MTOC), granule polarization to the IS, and degranulation. Here, we describe confocal live-cell imaging methods for the analysis of these processes at the immunological synapse, with a focus on mechanisms of cancer cell resistance facilitating escape from NK cell cytotoxicity.

Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.

BACKGROUND: Antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after mRNA or adenoviral vector-based vaccines is weak in kidney transplant (KT) patients. However, few studies have focused on humoral response after inactivated virus-based vaccines in KT. Here, we compare antibody response following vaccination with inactivated virus (CoronaVac®) and BNT162b2 mRNA. METHODS: A national multicentre cross-sectional study was conducted. The study group was composed of patients from all KT centres in Uruguay, vaccinated between 1 and 31 May 2021 (CoronaVac®, n = 245 and BNT162b2, n = 39). The control group was constituted of 82 healthy individuals. Participants had no prior confirmed coronavirus disease 2019 (COVID-19) test. Blood samples were collected between 30 and 40 days after the second dose. Serum-specific immunoglobulin G (IgG) antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein were determined using the COVID-19 IgG QUANT ELISA Kit. RESULTS: Only 29% of KT recipients showed seroconversion (36.5% BNT162b2, 27.8% inactivated virus, P = 0.248) in comparison with 100% in healthy control with either vaccine. Antibody levels against RBD were higher with BNT162b mRNA than with inactivated virus [median (interquartile range) 173 (73-554) and 29 (11-70) binding antibody units (BAU)/mL, P < 0.034] in KT and 10 times lower than healthy control [inactivated virus: 308 (209-335) and BNT162b2: 2638 (2608-3808) BAU/mL, P < 0.034]. In multivariate analysis, variables associated with negative humoral response were age, triple immunosuppression, estimated glomerular filtration rate and time post-KT. CONCLUSION: Seroconversion was low in KT patients after vaccination with both platforms. Antibody levels against SARS-CoV-2 were lower with inactivated virus than BNT162b mRNA. These findings support the need for strategies to improve immunogenicity in KT recipients after two doses of either vaccine.

Satisfacción con las clases virtuales de adolescentes en Costa Rica / Teens' satisfaction with virtual classes in Costa Rica

Resumen Durante la pandemia por COVID-19, el sistema educativo costarricense ha adaptado las clases a una modalidad virtual; además, hay evidencia de brechas tecnológicas, digitales y educativas entre la población estudiantil. El objetivo del presente estudio fue identificar las características sociodemográficas, psicosociales, tecnológicas y digitales asociadas al nivel de satisfacción de adolescentes en Costa Rica con las clases virtuales durante la pandemia. Se empleó un diseño transversal y se aplicó un cuestionario en línea a adolescentes de entre 14 y 18 años (n = 128, 63.3% mujeres, Medad = 16.2, DEedad = 0.593) de colegios públicos y privados. Se encontró que solo el apoyo percibido, la autoeficacia hacia las clases virtuales y el nivel socioeconómico (NSE) se correlacionan significativamente (de forma positiva) con la satisfacción con las clases virtuales. Además, se halló que un modelo que explica la variabilidad en la satisfacción a partir del apoyo percibido y la autoeficacia hacia las clases virtuales es mejor que uno que también incluya el NSE como variable predictora. Los resultados muestran indicios sobre la forma de hacer más satisfactoria la experiencia de las personas adolescentes en el contexto de clases virtuales.

Abstract During the COVID-19 pandemic the Costa Rican educational system has adapted classes to a virtual model; fur- thermore, evidence has supported the existence of technological, digital, and educational gaps among students. The aim of the present study is to identify sociodemographic, psychosocial, technological, and digital characteristics associated with the adolescents' satisfaction with virtual classes during the pandemic. Using a cross-sectional design, an online questionnaire was filled out by 14- to 18-year-old adolescents (n = 128, 63.3% girls, Mage = 16.2, SDage = 0.593) from public and private high schools. Results showed that only perceived support, self-efficacy, and socioeconomic status (SES) were significantly (positively) correlated with satisfaction with virtual classes. Also, it was found that a model that accounts for satisfaction in terms of perceived support and self-efficacy is better than one that includes SES as another explanatory variable. These results show some insights for making virtual learning a more satisfying experience for adolescents.

Autologous Dendritic Cells in Combination With Chemotherapy Restore Responsiveness of T Cells in Breast Cancer Patients: A Single-Arm Phase I/II Trial.

Introduction: Animal studies and preclinical studies in cancer patients suggest that the induction of immunogenic cell death (ICD) by neoadjuvant chemotherapy with doxorubicin and cyclophosphamide (NAC-AC) recovers the functional performance of the immune system. This could favor immunotherapy schemes such as the administration of antigen-free autologous dendritic cells (DCs) in combination with NAC-AC to profit as cryptic vaccine immunogenicity of treated tumors. Objective: To explore the safety and immunogenicity of autologous antigen-free DCs administered to breast cancer patients (BCPs) in combination with NAC-AC. Materials and Methods: A phase I/II cohort clinical trial was performed with 20 BCPs treated with NAC-AC [nine who received DCs and 11 who did not (control group)]. The occurrence of adverse effects and the functional performance of lymphocytes from BCPs before and after four cycles of NAC-AC receiving DCs or not were assessed using flow cytometry and compared with that from healthy donors (HDs). Flow cytometry analysis using manual and automated algorithms led us to examine functional performance and frequency of different lymphocyte compartments in response to a stimulus in vitro. This study was registered at clinicaltrials.gov (NCT03450044). Results: No grade II or higher adverse effects were observed associated with the transfer of DCs to patients during NAC-AC. Interestingly, in response to the in vitro stimulation, deficient phosphorylation of Zap70 and AKT proteins observed before chemotherapy in most patients' CD4 T cells significantly recovered after NAC-AC only in patients who received DCs. Conclusions: The transfer of autologous DCs in combination with NAC-AC in BCPs is a safe procedure. That, in BCPs, the administration of DCs in combination with NAC-AC favors the recovery of the functional capacity of T cells suggests that this combination may potentiate the adjuvant effect of ICD induced by NAC-AC on T cells and, hence, potentiate the immunogenicity of tumors as cryptic vaccines.

Dual Targeting of Stromal Cell Support and Leukemic Cell Growth by a Peptidic PKC Inhibitor Shows Effectiveness against B-ALL.

Mesenchymal stem cells (MSC) favour a scenario where leukemic cells survive. The protein kinase C (PKC) is essential to confer MSC support to leukemic cells and may be responsible for the intrinsic leukemic cell growth. Here we have evaluated the capacity of a chimeric peptide (HKPS), directed against classical PKC isoforms, to inhibit leukemic cell growth. HKPS was able to strongly inhibit viability of different leukemic cell lines, while control HK and PS peptides had no effect. Further testing showed that 30% of primary samples from paediatric B-cell acute lymphoblastic leukaemia (B-ALL) were also strongly affected by HKPS. We showed that HKPS disrupted the supportive effect of MSC that promote leukemic cell survival. Interestingly, ICAM-1 and VLA-5 expression increased in MSC during the co-cultures with B-ALL cells, and we found that HKPS inhibited the interaction between MSC and B-ALL cells due to a reduction in the expression of these adhesion molecules. Of note, the susceptibility of B-ALL cells to dexamethasone increased when MSC were treated with HKPS. These results show the relevance of these molecular interactions in the leukemic niche. The use of HKPS may be a new strategy to disrupt intercellular communications, increasing susceptibility to therapy, and at the same time, directly affecting the growth of PKC-dependent leukemic cells.

CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis.

BACKGROUND: Most carcinomas are composed of heterogeneous populations of tumor cells with distinct and apparently stable phenotypic characteristics. METHODS: Using an in vitro model of carcinogenesis we aimed at experimentally elucidating the significance of heterogeneity in the expression of CD24, a marker frequently overexpressed in various cancers and correlated with poor prognosis. RESULTS: We show that CD24Neg and CD24Pos cells issued from the same tumorigenic cell line display striking differences in stem-related properties, expression of epithelial-mesenchymal transition/mesenchymal-epithelial transition markers, and tumorigenic capacity. Indeed, while CD24Neg cells were as tumorigenic as the parental cell line, CD24Pos cells, although unable to form tumors, were unexpectedly more mesenchymal, displayed enhanced stemness-related properties, and expressed a proinflammatory signature. CONCLUSION: Our findings support the view that acquisition of stem-like cell, CD24-associated, attributes like migration, invasion, and plasticity by a tumor subpopulation is not necessarily related to local tumor growth but may be required for escaping the niche and colonizing distant sites.

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line.

BACKGROUND: There is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype (SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines. METHODS: The MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231. RESULTS: Our results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features. CONCLUSIONS: This study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.

The senescent microenvironment promotes the emergence of heterogeneous cancer stem-like cells.

There is a well-established association between aging and the onset of metastasis. Although the mechanisms through which age impinges upon the malignant phenotype remain uncharacterized, the role of a senescent microenvironment has been emphasized. We reported previously that human epithelial cells that undergo telomere-driven chromosome instability (T-CIN) display global microRNA (miR) deregulation and develop migration and invasion capacities. Here, we show that post-crisis cells are not able to form tumors unless a senescent microenvironment is provided. The characterization of cell lines established from such tumors revealed that these cells have acquired cell autonomous tumorigenicity, giving rise to heterogeneous tumors. Further experiments demonstrate that explanted cells, while displaying differences in cell differentiation markers, are all endowed of enhanced stem cell properties including self-renewal and multilineage differentiation capacity. Treatments of T-CIN+ cells with senescence-conditioned media induce sphere formation exclusively in cells with senescence-associated tumorigenicity, a capacity that depends on miR-145 repression. These results indicate that the senescent microenvironment, while promoting further transdifferentiations in cells with genome instability, is able to propel the progression of premalignant cells towards a malignant, cell stem-like state.

Necesidad de modificación normativa de los procesos contenciosos administrativos

Menciona que debe tenerse como principio general que todos los derechos vulnerados por actos del administrador son susceptibles de producir una acción contenciosa administrativa, entanto en cuanto ese derecho sea administrativo, esto se rige bajo el derecho administrativo...