CD28 costimulation augments CAR signaling in NK cells via the LCK/CD3Z/ZAP70 signaling axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3Z creates a platform that recruits critical kinases, such as LCK and ZAP70, initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy.

"It changed everything we do": A mixed methods study of youth and parent experiences with a pilot exercise education intervention following new diagnosis of type 1 diabetes.

AIMS: This pilot study delivered a comprehensive exercise education intervention to youth with new-onset type 1 diabetes (T1D) and their parents to increase knowledge and confidence with physical activity (PA) shortly after diagnosis. METHODS: Youth initiated continuous glucose monitoring (CGM) and PA trackers within 1 month of diagnosis. Youth and their parents received the 4-session intervention over 12 months. Participants completed self-report questionnaires at baseline, 6- and 12-months. Surveys were analyzed using linear mixed effects models. Semi-structured interviews and focus groups explored experiences with the exercise education intervention. Groups and interviews were audio-recorded, transcribed, and analyzed using content analysis. RESULTS: A total of 16 parents (aged 46 ± 7 years; 88 % female; 67 % non-Hispanic White) and 17 youth (aged 14 ± 2 years; 41 % female; 65 % non-Hispanic White) participated. Worry about hypoglycemia did not worsen throughout the study duration. Parents and youth reported increased knowledge and confidence in managing T1D safely and preventing hypoglycemia during PA following receiving the tailored exercise education intervention. CONCLUSION: This study assessed a novel structured exercise education program for youth and their parents shortly following T1D diagnosis. These results support the broad translation and acceptability of a structured exercise education program in new-onset T1D.

Hysteresis reveals a happiness bias effect in dynamic emotion recognition from ambiguous biological motion.

Considering the nonlinear dynamic nature of emotion recognition, it is believed to be strongly dependent on temporal context. This can be investigated by resorting to the phenomenon of hysteresis, which features a form of serial dependence, entailed by continuous temporal stimulus trajectories. Under positive hysteresis, the percept remains stable in visual memory (persistence) while in negative hysteresis, it shifts earlier (adaptation) to the opposite interpretation. Here, we asked whether positive or negative hysteresis occurs in emotion recognition of inherently ambiguous biological motion, while testing for the controversial debate of a negative versus positive emotional bias. Participants (n = 22) performed a psychophysical experiment in which they were asked to judge stimulus transitions between two emotions, happiness and sadness, from an actor database, and report perceived emotion across time, from one emotion to the opposite as physical cues were continuously changing. Our results reveal perceptual hysteresis in ambiguous emotion recognition, with positive hysteresis (visual persistence) predominating. However, negative hysteresis (adaptation/fatigue) was also observed in particular in the direction from sadness to happiness. This demonstrates a positive (happiness) bias in emotion recognition in ambiguous biological motion recognition. Finally, the interplay between positive and negative hysteresis suggests an underlying competition between visual persistence and adaptation mechanisms during ambiguous emotion recognition.

Programa de inoculación de estrés para el manejo del malestar emocional asociado a la terapia de células CAR-T: caso clínico / Stress inoculation program for the management of emotional distress associated with CART Cell Therapy: A case study

Objetivo: se presenta un caso clínico de una paciente con linfoma B difuso candidata a recibir tratamiento de células CART, con la que se utiliza un programa de inoculación de estrés debido a las características de la enfermedad y del tratamiento médico, además de la incertidumbre asociada. Metodología: programa de diez sesiones de intervención que se divide en tres fases: conceptualización, que incluye el modelo teórico de estrés y de intolerancia a la incertidumbre; adquisición de estrategias y ensayo que incorpora técnicas de desactivación y cognitivas, planificación de actividades y entrenamiento en habilidades de comunicación; consolidación. Resultados: se evidencia una reducción del malestar emocional, de la sintomatología de ansiedad y bajo estado de ánimo, así como de los niveles de fatiga. Respecto a las estrategias de afrontamiento, tras la intervención psicológica se mantiene el estilo de preocupación ansiosa, aunque se observa un mejor uso de recursos asociados al afrontamiento de espíritu de lucha. Conclusión: la aplicación sistemática de programas para el manejo del estrés puede suponer una estrategia útil y efectiva para la mejora de la salud mental de los pacientes que van a someterse a tratamiento de células CART (AU)

Objective: A case report is presented of a patient with diffuse B lymphoma who is a candidate for CART cell therapy using a stress inoculation program due to the characteristics of the disease and medical treatment and the associated uncertainty. Methodology: psychological program intervention with 10 sessions, divided into three phases: Conceptualization, including the theoretical model of stress and uncertainty intolerance; strategy acquisition and rehearsal, incorporating deactivation and cognitive techniques, activity planning and communication skills training; consolidation. Results: there was a reduction in emotional distress, anxiety symptoms and mood, as well as fatigue levels. In terms of coping strategies, after the psychological intervention, the anxious worrying style is maintained, although a better use of the resources associated with coping with a fighting spirit is observed. Conclusion: Systematic application of stress management programs can be a useful and effective strategy for improving the mental health of patients undergoing CART cell treatment (AU)

KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir.

Human immunodeficiency virus (HIV) infection induces immunological dysfunction, which limits the elimination of HIV-infected cells during treated infection. Identifying and targeting dysfunctional immune cells might help accelerate the purging of the persistent viral reservoir. Here, we show that chronic HIV infection increases natural killer (NK) cell populations expressing the negative immune regulator KLRG1, both in peripheral blood and lymph nodes. Antiretroviral treatment (ART) does not reestablish these functionally impaired NK populations, and the expression of KLRG1 correlates with active HIV transcription. Targeting KLRG1 with specific antibodies significantly restores the capacity of NK cells to kill HIV-infected cells, reactivates latent HIV present in CD4+ T cells co-expressing KLRG1, and reduces the intact HIV genomes in samples from ART-treated individuals. Our data support the potential use of immunotherapy against the KLRG1 receptor to impact the viral reservoir during HIV persistence.

La eficacia de la terapia cognitivo-conductual para el duelo prolongado / Efficacy of cognitive-behavioral therapy for prolonged grief

Antecedentes: El Duelo Prolongado (DP) es una forma patológica del duelo dados los síntomas patentes y el tiempo que perduran, generando malestar y limitando la funcionalidad psicosocial. Durante años se ha empleado la Terapia Cognitivo-Conductual (TCC) para el tratamiento del DP, pero se sugiere que la activación conductual no específica es suficiente, se hallan fallos metodológicos en las muestras de TCC, o se cuestiona si es necesaria la exposición. Objetivo: Analizar si la TCC es realmente eficaz para mejorar el DP en adultos. Método: Se realizó una revisión sistemática mediante la búsqueda en las bases de datos Medline, PsycINFO, PubMed, Scopus y Web of Science desde junio de 2022 hasta febrero de 2023, así como mediante una comunicación personal. Se identificaron 9 estudios que cumplían los criterios de inclusión y exclusión establecidos. Resultados: Se comparan las evaluaciones basales, del postratamiento y seguimiento. Todos los estudios reflejan mejorías superiores en la condición de TCC frente a otras modalidades terapéuticas o frente a la propia TCC sin exposición, a excepción de uno que establece igualdad de eficacia con Eye Movement Desensitization and Reprocessing (EMDR). Se analiza el riesgo de sesgo siguiendo los criterios Cochrane, presentando una adecuada calidad metodológica; el nivel de evidencia científica, 1+; y el grado de recomendación, A. Conclusiones: Los resultados respaldan la eficacia de la TCC para el tratamiento del DP en la población adulta, aunque es fundamental realizar nuevos estudios que fortalezcan esta evidencia. Hasta entonces, se recomienda su aplicación a quienes padezcan DP (AU)

Background: Prolonged Grief (PG) is a pathological form of bereavement given the overt symptoms and the period of time they endure, generating distress and limiting psychosocial functioning. For years, Cognitive Behavioral-Therapy (CBT) has been used for the treatment of PG. Nonetheless, it is suggested that non-specific behavioral activation could be sufficient, methodological problems are found in CBT samples, or it is questioned whether the exposure component is necessary or not. Objective: To analyze whether CBT is really effective to improve PG among the adult population. Methods: A systematic review was conducted through Medline, PsycINFO, PubMed, Scopus and Web of Science databases from June 2022 to February 2023, as well as by means of personal communication. 9 studies that met the established inclusion and exclusion criteria were identified. Results: Baseline post-treatment and follow-up assessments are compared. All studies reflect superior improvements in the CBT condition compared to other therapeutic modalities or compared to CBT without exposure, excluding one study that establishes equal efficacy with Eye Movement Desensitization and Reprocessing (EMDR). The risk of bias was analyzed according to Cochrane criteria, presenting an adequate methodological quality; the level of scientific evidence, 1+; and the grade of recommendation, A. Conclusions: The results support the efficacy of CBT for the treatment of PG in the adult population. Nevertheless, it is essential to conduct new studies that strengthen this evidence. Until then, it is recommended its application among those suffering from PG (AU)

Activated B cells suppress T-cell function through metabolic competition.

BACKGROUND: B cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood. METHODS: We investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing. RESULTS: Here we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell-cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy. CONCLUSIONS: We have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

A phase two study of high dose blinatumomab in Richter's syndrome.

Richter's Syndrome (RS) is an aggressive transformation of CLL, usually clonally-related diffuse large B-cell lymphoma (DLBCL), characterized by frequent TP53 mutations, intrinsic chemoresistance and poor survival. TP53-independent treatments are needed. We conducted a single center, phase 2, investigator-initiated study of high dose blinatumomab (maximum 112 mcg/d after initial, weekly dose escalation), NCT03121534, given for an 8-week induction and 4-week consolidation cycle. Responses were assessed by Lugano 2014 criteria. Serial multi-parameter flow cytometry from blood was performed to identify patient-specific biomarkers for response. Nine patients were treated. Patients had received a median of 4 and 2 prior therapies for CLL and RS, respectively. Five of 9 had del(17p) and 100% had complex karyotype. Four patients had reduction in nodal disease, including one durable complete response lasting >1 y. Treatment was well tolerated, with no grade >3 cytokine release syndrome and 1 case of grade 3, reversible neurotoxicity. Immunophenotyping demonstrated the majority of patients expressed multiple immune checkpoints, especially PD1, TIM3 and TIGIT. The patient who achieved CR had the lowest levels of immune checkpoint expression. Simultaneous targeting with immune checkpoint blockade, especially PD1 inhibition, which has already demonstrated single-agent efficacy in RS, could achieve synergistic killing and enhance outcomes.

'Much more convenient, just as effective': Experiences of starting continuous glucose monitoring remotely following Type 1 diabetes diagnosis.

AIM: Initiating continuous glucose monitoring (CGM) shortly after Type 1 diabetes diagnosis has glycaemic and quality of life benefits for youth with Type 1 diabetes and their families. The SARS-CoV-2 pandemic led to a rapid shift to virtual delivery of CGM initiation visits. We aimed to understand parents' experiences receiving virtual care to initiate CGM within 30 days of diagnosis. METHODS: We held focus groups and interviews using a semi-structured interview guide with parents of youth who initiated CGM over telehealth within 30 days of diagnosis during the SARS-CoV-2 pandemic. Questions aimed to explore experiences of starting CGM virtually. Groups and interviews were audio-recorded, transcribed and analysed using thematic analysis. RESULTS: Participants were 16 English-speaking parents (age 43 ± 6 years; 63% female) of 15 youth (age 9 ± 4 years; 47% female; 47% non-Hispanic White, 20% Hispanic, 13% Asian, 7% Black, 13% other). They described multiple benefits of the virtual visit including convenient access to high-quality care; integrating Type 1 diabetes care into daily life; and being in the comfort of home. A minority experienced challenges with virtual care delivery; most preferred the virtual format. Participants expressed that clinics should offer a choice of virtual or in-person to families initiating CGM in the future. CONCLUSION: Most parents appreciated receiving CGM initiation education via telehealth and felt it should be an option offered to all families. Further efforts can continue to enhance CGM initiation teaching virtually to address identified barriers.

Impact of previous cardiac function status assessed by echocardiography on the outcome of COVID-19.

More than 91,000 fatalities due to Coronavirus Disease 2019 (COVID-19) have occurred in Spain. Several factors are associated with increased mortality in this disease, including cardiovascular risk factors (CVRF). However, information on the cardiac function of patients prior to the onset of COVID-19 is scarce and the potential impact it may have is uncertain. The aim of the EchoVID study was to describe the potential association between CVRF and cardiac function status prior to SARS-CoV-2 infection and in-hospital mortality. We studied clinical characteristics and cardiac function of patients admitted during the first wave of COVID-19. All patients had a transthoracic echocardiogram performed in the previous 12 months prior to diagnosis; conventional systolic and diastolic function parameters were analyzed. Logistic regression analysis was performed to identify predictors of in-hospital mortality. We included 296 individuals. Median age was higher in the group of patients who died (81.0 vs 76.1 years; p = 0.007). No significant differences were found in CVRF. Survivors were more frequently receiving anticoagulation therapy (52.9% vs 70.8%; p = 0.003). LVEF, although preserved on average in both groups, was significantly lower in the group of deceased patients (56.9% vs 61.1%; p = 0.017). Average E/e' ratio was higher in the deceased group (11.1 vs 10.1; p = 0.049). Five variables were found to be independently associated with in-hospital mortality due to COVID-19: Age, male gender, LVEF, E/e' ratio and anticoagulation therapy. A model including these variables had an area under the ROC curve of 0.756 (CI 0.669-0.843). The echocardiographic variables included in the model significantly improved the discriminative power, compared to a model including only demographic data. Left ventricular ejection fraction and E/e' ratio prior to SARS-CoV-2 infection are two easily-obtained echocardiographic parameters that provide additional prognostic information over clinical factors when assessing patients admitted for SARS-CoV-2 infection.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

Candidalysins Are a New Family of Cytolytic Fungal Peptide Toxins.

Candidalysin is the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is secreted by Candida albicans and is critical for driving infection and host immune responses in several model systems. However, Candida infections are also caused by non-C. albicans species. Here, we identify and characterize orthologs of C. albicans candidalysin in C. dubliniensis and C. tropicalis. The candidalysins have different amino acid sequences, are amphipathic, and adopt a predominantly α-helical secondary structure in solution. Comparative functional analysis demonstrates that each candidalysin causes epithelial damage and calcium influx and activates intracellular signaling pathways and cytokine secretion. Importantly, C. dubliniensis and C. tropicalis candidalysins have higher damaging and activation potential than C. albicans candidalysin and exhibit more rapid membrane binding and disruption, although both fungal species cause less damage to epithelial cells than C. albicans. This study identifies the first family of peptide cytolysins in human-pathogenic fungi. IMPORTANCE Pathogenic fungi kill an estimated 1.5 million people every year. Recently, we discovered that the fungal pathogen Candida albicans secretes a peptide toxin called candidalysin during mucosal infection. Candidalysin causes damage to host cells, a process that supports disease progression. However, fungal infections are also caused by Candida species other than C. albicans. In this work, we identify and characterize two additional candidalysin toxins present in the related fungal pathogens C. dubliniensis and C. tropicalis. While the three candidalysins have different amino acid sequences, all three toxins are α-helical and amphipathic. Notably, the candidalysins from C. dubliniensis and C. tropicalis are more potent at inducing cell damage, calcium influx, mitogen-activated protein kinase signaling, and cytokine responses than C. albicans candidalysin, with the C. dubliniensis candidalysin having the most rapid membrane binding kinetics. These observations identify the candidalysins as the first family of peptide toxins in human-pathogenic fungi.

Fisioterapia respiratoria en pacientes adultos post-COVID-19: revisión sistemática de la literatura / Respiratory physiotherapy in post-acute COVID-19 adult patients: Systematic review of literature

Introducción: los pacientes con SARS-CoV-2 presentan signos y síntomas que involucran principalmente el sistema respiratorio. Las secuelas son consecuencia de un deterioro de la calidad de vida, neumonía, fatiga, disnea y dolor articular. Objetivo: tener el sustento científico que permita evidenciar la importancia de la fisioterapia respiratoria y sus efectos sobre los pacientes adultos post-COVID-19 de fase aguda. Material y métodos: se hizo una revisión sistemática de la literatura en cuatro bases de datos (Scopus, Web of Science, PubMed y ScienceDirect). La búsqueda fue realizada en febrero de 2021 con un total de 1229 estudios. Finalmente, se incluyeron cinco estudios que cumplieron con los criterios de elegibilidad: dos ensayos clínicos, dos reportes de caso y un estudio transversal. La calidad metodológica de las publicaciones fue evaluada. Resultados: el entrenamiento de la musculatura respiratoria, las respiraciones dirigidas y el fortalecimiento general dan datos significativos en la mejora de la funcionalidad. La evidencia demuestra que hay efectos positivos de la fisioterapia respiratoria en pacientes adultos post-COVID-19, pues aumenta la resistencia al ejercicio, disminuye la fatiga, se reduce la disnea, mejora la funcionalidad y la calidad de vida. Conclusiones: es necesario que se desarrollen más ensayos clínicos aleatorizados y estudios de grupos de menor rango de edad y con enfoques individualizados.

Background: Patients with SARS-CoV-2 present signs and symptoms that primarily involve the respiratory system. The sequelae result in impaired quality of life, pneumonia, dyspnea, fatigue, and joint pain.

Testing the effectiveness of community-engaged citizen science to promote physical activity, foster healthier neighborhood environments, and advance health equity in vulnerable communities: The Steps for Change randomized controlled trial design and methods.

While low-income midlife and older adults are disproportionately affected by non-communicable diseases that can be alleviated by regular physical activity, few physical activity programs have been developed specifically with their needs in mind. Those programs that are available typically do not address the recognized local environmental factors that can impact physical activity. The specific aim of the Steps for Change cluster-randomized controlled trial is to compare systematically the initial (one-year) and sustained (two-year) multi-level impacts of an evidence-based person-level physical activity intervention (Active Living Every Day [ALED] and age-relevant health education information), versus the ALED program in combination with a novel neighborhood-level citizen science intervention called Our Voice. The study sample (N = 300) consists of insufficiently active adults ages 40 years and over living in or around affordable senior public housing settings. Major study assessments occur at baseline, 12, and 24 months. The primary outcome is 12-month change in walking, and secondary outcomes include other forms of physical activity, assessed via validated self-report measures supported by accelerometry, and physical function and well-being variables. Additional intervention impacts are assessed at 24 months. Potential mediators and moderators of intervention success will be explored to better determine which subgroups do best with which type of intervention. Here we present the study design and methods, including recruitment strategies and yields. TRIAL REGISTRATION: clinicaltrial.gov Identifier = NCT03041415.

Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Favipiravir-resistant influenza A virus shows potential for transmission.

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies.

PURPOSE: Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. EXPERIMENTAL DESIGN: We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated in vitro and in vivo. RESULTS: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. CONCLUSIONS: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.