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Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.
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Linfocitos B/inmunología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Estructuras Linfoides Terciarias/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/cirugía , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Células Clonales/citología , Células Clonales/inmunología , Células Clonales/metabolismo , Células Dendríticas Foliculares/citología , Células Dendríticas Foliculares/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Memoria Inmunológica/inmunología , Espectrometría de Masas , Melanoma/patología , Melanoma/cirugía , Metástasis de la Neoplasia/genética , Fenotipo , Pronóstico , RNA-Seq , Receptores Inmunológicos/inmunología , Análisis de la Célula Individual , Linfocitos T/citología , Linfocitos T/inmunología , TranscriptomaRESUMEN
Patients with B-lineage acute lymphoblastic leukemia (ALL) are at high-risk for relapse after allogeneic hematopoietic cell transplantation (HCT). We conducted a single-center phase 2 study evaluating the feasibility of 4 cycles of blinatumomab administered every 3 months during the first year after HCT in an effort to mitigate relapse in high-risk ALL patients. Twenty-one of 23 enrolled patients received at least 1 cycle of blinatumomab and were included in the analysis. The median time from HCT to the first cycle of blinatumomab was 78 days (range, 44 to 105). Twelve patients (57%) completed all 4 treatment cycles. Neutropenia was the only grade 4 adverse event (19%). Rates of cytokine release (5% G1) and neurotoxicity (5% G2) were minimal. The cumulative incidence of acute graft-versus-host disease (GVHD) grades 2 to 4 and 3 to 4 were 33% and 5%, respectively; 2 cases of mild (10%) and 1 case of moderate (5%) chronic GVHD were noted. With a median follow-up of 14.3 months, the 1-year overall survival (OS), progression-free survival (PFS), and nonrelapse mortality (NRM) rates were 85%, 71%, and 0%, respectively. In a matched analysis with a contemporary cohort of 57 patients, we found no significant difference between groups regarding blinatumomab's efficacy. Correlative studies of baseline and posttreatment samples identified patients with specific T-cell profiles as "responders" or "nonresponders" to therapy. Responders had higher proportions of effector memory CD8 T-cell subsets. Nonresponders were T-cell deficient and expressed more inhibitory checkpoint molecules, including T-cell immunoglobulin and mucin domain 3 (TIM3). We found that blinatumomab postallogeneic HCT is feasible, and its benefit is dependent on the immune milieu at time of treatment. This paper is posted on ClinicalTrials.gov, study ID: NCT02807883.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Enfermedad Aguda , Anticuerpos Biespecíficos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , RecurrenciaRESUMEN
BACKGROUND: Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in B-cell cancers. However, CAR T cells can induce substantial toxic effects, and the manufacture of the cells is complex. Natural killer (NK) cells that have been modified to express an anti-CD19 CAR have the potential to overcome these limitations. METHODS: In this phase 1 and 2 trial, we administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1×105, 1×106, or 1×107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy. RESULTS: The administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline. The maximum tolerated dose was not reached. Of the 11 patients who were treated, 8 (73%) had a response; of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter's transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months. CONCLUSIONS: Among 11 patients with relapsed or refractory CD19-positive cancers, a majority had a response to treatment with CAR-NK cells without the development of major toxic effects. (Funded by the M.D. Anderson Cancer Center CLL and Lymphoma Moonshot and the National Institutes of Health; ClinicalTrials.gov number, NCT03056339.).
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Antígenos CD19 , Células Asesinas Naturales/trasplante , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma no Hodgkin/terapia , Receptores Quiméricos de Antígenos/antagonistas & inhibidores , Anciano , Aloinjertos , Tratamiento Basado en Trasplante de Células y Tejidos , Femenino , Sangre Fetal , Vectores Genéticos , Humanos , Células Asesinas Naturales/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma no Hodgkin/inmunología , Masculino , Persona de Mediana Edad , Inducción de Remisión/métodos , Retroviridae/genética , Acondicionamiento PretrasplanteRESUMEN
Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.
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Sangre Fetal/citología , Inmunoterapia Adoptiva , Interleucina-15/genética , Células Asesinas Naturales/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Aerobiosis , Animales , Antígenos CD19/inmunología , Linfoma de Burkitt/patología , Linfoma de Burkitt/terapia , Sistemas CRISPR-Cas , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/trasplante , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores Quiméricos de Antígenos , Transducción de Señal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Signal intensity measured in a mass cytometry (CyTOF) channel can often be affected by the neighboring channels due to technological limitations. Such signal artifacts are known as spillover effects and can substantially limit the accuracy of cell population clustering. Current approaches reduce these effects by using additional beads for normalization purposes known as single-stained controls. While effective in compensating for spillover effects, incorporating single-stained controls can be costly and require customized panel design. This is especially evident when executing large-scale immune profiling studies. We present a novel statistical method, named CytoSpill that independently quantifies and compensates the spillover effects in CyTOF data without requiring the use of single-stained controls. Our method utilizes knowledge-guided modeling and statistical techniques, such as finite mixture modeling and sequential quadratic programming, to achieve optimal error correction. We evaluated our method using five publicly available CyTOF datasets obtained from human peripheral blood mononuclear cells (PBMCs), C57BL/6J mouse bone marrow, healthy human bone marrow, chronic lymphocytic leukemia patient, and healthy human cord blood samples. In the PBMCs with known ground truth, our method achieved comparable results to experiments that incorporated single-stained controls. In datasets without ground-truth, our method not only reduced spillover on likely affected markers, but also led to the discovery of potentially novel subpopulations expressing functionally meaningful, cluster-specific markers. CytoSpill (developed in R) will greatly enhance the execution of large-scale cellular profiling of tumor immune microenvironment, development of novel immunotherapy, and the discovery of immune-specific biomarkers. The implementation of our method can be found at https://github.com/KChen-lab/CytoSpill.git.
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Leucocitos Mononucleares , Animales , Biomarcadores , Análisis por Conglomerados , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.
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Linfocitos T CD4-Positivos/inmunología , Regulación Leucémica de la Expresión Génica , Memoria Inmunológica , Gripe Humana/prevención & control , Leucemia Mieloide Aguda/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Antibióticos Antineoplásicos/farmacología , Anticuerpos/farmacología , Transporte Biológico , Antígenos CD4/genética , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/inmunología , Daunorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Humanos , Inmunofenotipificación , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/inmunología , Gripe Humana/virología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/virología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/crecimiento & desarrollo , Orthomyxoviridae/inmunología , Rodaminas/metabolismo , Rodaminas/farmacología , Transducción de Señal , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/patologíaRESUMEN
Cord blood (CB) offers a number of advantages over other sources of hematopoietic stem cells, including a lower rate of chronic graft-versus-host disease (cGVHD) in the presence of increased HLA disparity. Recent research in experimental models of autoimmunity and in patients with autoimmune or alloimmune disorders has identified a functional group of interleukin-10 (IL-10)-producing regulatory B cells (Bregs) that negatively regulate T-cell immune responses. At present, however, there is no consensus on the phenotypic signature of Bregs, and their prevalence and functional characteristics in CB remain unclear. Here, we demonstrate that CB contains an abundance of B cells with immunoregulatory function. Bregs were identified in both the naive and transitional B-cell compartments and suppressed T-cell proliferation and effector function through IL-10 production as well as cell-to-cell contact involving CTLA-4. We further show that the suppressive capacity of CB-derived Bregs can be potentiated through CD40L signaling, suggesting that inflammatory environments may induce their function. Finally, there was robust recovery of IL-10-producing Bregs in patients after CB transplantation, to higher frequencies and absolute numbers than seen in the peripheral blood of healthy donors or in patients before transplant. The reconstituting Bregs showed strong in vitro suppressive activity against allogeneic CD4(+) T cells, but were deficient in patients with cGVHD. Together, these findings identify a rich source of Bregs and suggest a protective role for CB-derived Bregs against cGVHD development in CB recipients. This advance could propel the development of Breg-based strategies to prevent or ameliorate this posttransplant complication.
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Linfocitos B Reguladores/inmunología , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Sangre Fetal/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/terapia , Interleucina-10/metabolismo , Activación de Linfocitos/inmunología , Adulto , Anciano , Linfocitos T CD4-Positivos/inmunología , Femenino , Enfermedad Injerto contra Huésped/etiología , Neoplasias Hematológicas/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Transducción de Señal , Adulto JovenRESUMEN
The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1 HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.
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Trasplante de Células Madre de Sangre del Cordón Umbilical , Genotipo , Antígenos HLA/genética , Neoplasias Hematológicas , Receptores KIR/genética , Donante no Emparentado , Adulto , Anciano , Aloinjertos , Supervivencia sin Enfermedad , Femenino , Técnicas de Genotipaje , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidad , Neoplasias Hematológicas/terapia , Humanos , Masculino , Persona de Mediana Edad , Tasa de SupervivenciaRESUMEN
Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.
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Traslado Adoptivo/normas , Esclerosis Amiotrófica Lateral/patología , Separación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Cultivo Primario de Células , Linfocitos T Reguladores/patología , Traslado Adoptivo/métodos , Esclerosis Amiotrófica Lateral/inmunología , Estudios de Casos y Controles , Separación Celular/métodos , Separación Celular/normas , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Adhesión a Directriz/normas , Humanos , Tolerancia Inmunológica , Interleucina-2/metabolismo , Cultivo Primario de Células/métodos , Cultivo Primario de Células/normas , Linfocitos T Reguladores/inmunologíaRESUMEN
Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy (ACT), have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA sequencing (RNA-seq) datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.
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Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy, have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA-seq datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.
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Relapse is the major cause of failure of high-dose chemotherapy (HDC) with autologous stem cell transplantation (ASCT) for B cell non-Hodgkin lymphomas (B-NHL). Improvement strategies include use in combination with effective immunotherapies. We hypothesized that the combination of rituximab/HDC/ASCT with expanded cord blood (CB)-derived natural killer (NK) cells is safe and active in B-NHL. Patients with B-NHL age 15 to 70 years and appropriate ASCT candidates were eligible for the study. The CB units were selected without considering HLA match with the recipient. The CB NK cells were expanded from day -19 to day -5. Treatment included rituximab on days -13 and -7, BEAM (carmustine/etoposide/cytarabine/melphalan) on days -13 to -7, lenalidomide on days -7 to -2, CB NK infusion (108/kg) on day -5, and ASCT (day 0). The primary endpoint was 30-day treatment-related mortality (TRM); secondary endpoints included relapse-free survival (RFS), overall survival (OS), and persistence of CB NK cells. We enrolled 20 patients. CB NK cells were expanded a median of 1552-fold with >98% purity and >96% viability. We saw no adverse events attributable to the CB NK cells and 0% 30-day TRM. At median follow-up of 47 months, the RFS and OS rates were 53% and 74%, respectively. CB NK cells were detectable in blood for 2 weeks, independent of HLA-mismatch status. CD16 expression in donor NK cells was correlated favorably with outcome, and homozygosity for the high-affinity CD16 variant (158 V/V) in CB, but not recipient, NK cells was correlated with better outcomes. Our data indicate that the combination of expanded and highly purified CB-derived NK cells with HDC/ASCT for B-NHL is safe. CD16 expression in donor NK cells, particularly if homozygous for the high-affinity CD16 variant, was correlated with better outcomes.
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Trasplante de Células Madre Hematopoyéticas , Linfoma de Células B , Humanos , Adolescente , Adulto Joven , Adulto , Persona de Mediana Edad , Anciano , Rituximab/uso terapéutico , Sangre Fetal , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Trasplante Autólogo , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/etiología , Células Asesinas NaturalesRESUMEN
Acute myeloid leukemia (AML) is the most common acute leukemia in adults. While induction chemotherapy leads to remission in most patients, a significant number will experience relapse. Therefore, there is a need for novel therapies that can improve remission rates in patients with relapsed and refractory AML. CD70 is the natural ligand for CD27 (a member of the TNF superfamily) and appears to be a promising therapeutic target. Consequently, there is considerable interest in developing chimeric antigen receptor (CAR) T-cell therapy products that can specifically target CD70 in various neoplasms, including AML. In this study, we employed routine diagnostic techniques, such as immunohistochemistry and flow cytometry, to investigate the expression of CD70 in bone marrow samples from treatment-naïve and relapsed AML patients after hypomethylating agents (HMA). Also, we evaluated the impact of HMA on CD70 expression and examined CD70 expression in various leukemic cell subsets and normal hematopoietic progenitors.
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There is a pressing need for allogeneic chimeric antigen receptor (CAR)-immune cell therapies that are safe, effective and affordable. We conducted a phase 1/2 trial of cord blood-derived natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor and interleukin-15 (CAR19/IL-15) in 37 patients with CD19+ B cell malignancies. The primary objectives were safety and efficacy, defined as day 30 overall response (OR). Secondary objectives included day 100 response, progression-free survival, overall survival and CAR19/IL-15 NK cell persistence. No notable toxicities such as cytokine release syndrome, neurotoxicity or graft-versus-host disease were observed. The day 30 and day 100 OR rates were 48.6% for both. The 1-year overall survival and progression-free survival were 68% and 32%, respectively. Patients who achieved OR had higher levels and longer persistence of CAR-NK cells. Receiving CAR-NK cells from a cord blood unit (CBU) with nucleated red blood cells ≤ 8 × 107 and a collection-to-cryopreservation time ≤ 24 h was the most significant predictor for superior outcome. NK cells from these optimal CBUs were highly functional and enriched in effector-related genes. In contrast, NK cells from suboptimal CBUs had upregulation of inflammation, hypoxia and cellular stress programs. Finally, using multiple mouse models, we confirmed the superior antitumor activity of CAR/IL-15 NK cells from optimal CBUs in vivo. These findings uncover new features of CAR-NK cell biology and underscore the importance of donor selection for allogeneic cell therapies. ClinicalTrials.gov identifier: NCT03056339 .
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Trasplante de Células Madre Hematopoyéticas , Neoplasias , Receptores Quiméricos de Antígenos , Animales , Ratones , Humanos , Receptores Quiméricos de Antígenos/genética , Interleucina-15 , Células Asesinas Naturales , Inmunoterapia Adoptiva/efectos adversos , Antígenos CD19 , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM.
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Neoplasias Encefálicas , Proteína delta de Unión al Potenciador CCAAT , Glioblastoma , Interleucinas , Células Asesinas Naturales , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Glioblastoma/inmunología , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/terapia , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/inmunología , Humanos , Animales , Ratones , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteína delta de Unión al Potenciador CCAAT/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-15/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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INTRODUCTION: During pregnancy, a progressive increase in serum triglyceride (TG) and cholesterol levels is observed whereas TG levels mostly remain <300 mg/dl. In women with genetic forms of hypertriglyceridemia, pregnancy may cause extremely elevated TG levels leading to potentially life-threatening pancreatitis attacks and chylomicronemia syndrome. The only safe medical treatment option during pregnancy is ω-3 fatty acids, which have moderate TG lowering effects. Therapeutic apheresis could be used as primary treatment approach during pregnancy. MATERIALS AND METHODS: We reported the effect of double filtration apheresis in one pregnant women with severe hypertriglyceridemia, therapeutic plasmapheresis and double filtration methods in the other severe hypertriglyceridemic pregnant woman; a 32-year-old pregnant woman (patient 1) with a history of hypertriglyceridemia-induced acute pancreatitis during pregnancy and a 30-year-old pregnant woman with extremely high TG levels (12,000 mg/dl) leading to chylomicronemia syndrome (patient 2). Medical nutrition therapy and ω-3 fatty acids were also provided. Double filtration apheresis (patient 1) and plasmapheresis + double filtration apheresis (patient 2) were used. RESULT AND CONCLUSION: When we calculated the TG levels before and after therapeutic apheresis, maximum decrease achieved with double filtration apheresis was 46.3 % for patient 1 and 37.3 % for patient 2. However, with plasmapheresis TG level declined by 72 % in patient 2. Plasmapheresis seemed to be more efficient to decrease TG levels. Iron deficiency anemia was the main complication apart from technical difficulties by lipemic obstruction of tubing system. Healthy babies were born. Delivery led to decreases in TG levels. It is concluded that during pregnancy therapeutic apheresis is an effective method to decrease extremely high TG levels and risks of its potentially life-threatening complications.
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Eliminación de Componentes Sanguíneos/métodos , Hipertrigliceridemia/complicaciones , Hipertrigliceridemia/terapia , Plasmaféresis , Complicaciones del Embarazo/terapia , Enfermedad Aguda , Adulto , Cesárea , Ácidos Grasos Omega-3/administración & dosificación , Femenino , Filtración/métodos , Edad Gestacional , Humanos , Masculino , Pancreatitis/etiología , Embarazo , Resultado del EmbarazoRESUMEN
A Bayesian feature allocation model (FAM) is presented for identifying cell subpopulations based on multiple samples of cell surface or intracellular marker expression level data obtained by cytometry by time of flight (CyTOF). Cell subpopulations are characterized by differences in marker expression patterns, and cells are clustered into subpopulations based on their observed expression levels. A model-based method is used to construct cell clusters within each sample by modeling subpopulations as latent features, using a finite Indian buffet process. Non-ignorable missing data due to technical artifacts in mass cytometry instruments are accounted for by defining a static missingship mechanism. In contrast with conventional cell clustering methods, which cluster observed marker expression levels separately for each sample, the FAM-based method can be applied simultaneously to multiple samples, and also identify important cell subpopulations likely to be otherwise missed. The proposed FAM-based method is applied to jointly analyse three CyTOF datasets to study natural killer (NK) cells. Because the subpopulations identified by the FAM may define novel NK cell subsets, this statistical analysis may provide useful information about the biology of NK cells and their potential role in cancer immunotherapy which may lead, in turn, to development of improved NK cell therapies.
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Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.
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Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Citocinas/metabolismo , Línea Celular Tumoral , Células Asesinas Naturales , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.