RESUMEN
BACKGROUND: Outcomes are poor for patients with large B-cell lymphoma who relapse after CD19-directed chimeric antigen receptor (CAR) T-cell therapy (CAR19). CD22 is a nearly universally expressed B-cell surface antigen and the efficacy of a CD22-directed CAR T-cell therapy (CAR22) in large B-cell lymphoma is unknown, which was what we aimed to examine in this study. METHODS: In this single centre, open-label, dose-escalation phase 1 trial, we intravenously administered CAR22 at two dose levels (1 million and 3 million CAR22-positive T cells per kg of bodyweight) to adult patients (aged ≥18 years) who relapsed after CAR19 or had CD19-negative large B-cell lymphoma. The primary endpoints were manufacturing feasibility, safety measured by the incidence and severity of adverse events and dose-limiting toxicities, and identification of the maximum tolerated dose (ie, the recommended phase 2 dose). This study is registered with ClinicalTrials.gov (NCT04088890) and is active, but closed for enrolment. FINDINGS: From Oct 17, 2019, to Oct 19, 2022, a total of 41 patients were assessed for eligibility; however, one patient withdrew. 40 patients underwent leukapheresis and 38 (95%) had CAR T-cell products manufactured successfully. The median age was 65 years (range 25-84), 17 (45%) were women, 32 (84%) had elevated pretreatment lactate dehydrogenase, 11 (29%) had refractory disease to all previous therapies, and patients had received a median of four lines of previous therapy (range 3-8). Of the 38 patients treated, 37 (97%) had relapsed after previous CAR19. The identified maximum tolerated dose was 1 million CAR T cells per kg. Of 29 patients who received the maximum tolerated dose, no patients developed a dose-limiting toxicity or grade 3 or higher cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, or immune effector cell-associated haemophagocytic lymphohistiocytosis-like syndrome. INTERPRETATION: This trial identifies CD22 as an immunotherapeutic target in large B-cell lymphoma and demonstrates the durable clinical activity of CAR22 in patients with disease progression after CAR19 therapy. Although these findings are promising, it is essential to recognise that this is a phase 1 dose-finding study. Further investigations are warranted to establish the long-term efficacy and to delineate the patient subgroups that will derive the most benefit from this therapeutic approach. FUNDING: National Cancer Institute, National Institutes of Health, Stanford Cancer Institute, Leukemia & Lymphoma Society, Parker Institute for Cancer Immunotherapy, Lymph & Co, and the European Hematology Association.
Asunto(s)
Antígenos CD19 , Inmunoterapia Adoptiva , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/efectos adversos , Linfoma de Células B Grandes Difuso/terapia , Linfoma de Células B Grandes Difuso/inmunología , Dosis Máxima Tolerada , Receptores Quiméricos de Antígenos/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Anciano de 80 o más AñosRESUMEN
The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Linfoma de Células B Grandes Difuso/terapia , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Ensayos Clínicos Fase I como Asunto , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Pronóstico , Inducción de RemisiónRESUMEN
Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Linfocitos/citología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunosupresores/farmacología , Linfocitos/enzimología , Ratones , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Isoformas de Proteínas , Transducción de Señal , Bazo/citología , Linfocitos T/citología , Linfocitos T/enzimologíaRESUMEN
The assessment of T-cell clonality by flow cytometry has long been suboptimal, relying on aberrant marker expression and/or intensity. The introduction of TRBC1 shows much promise for improving the diagnosis of T-cell neoplasms in the clinical flow laboratory. Most laboratories considering this marker already have existing panels designed for T-cell workups and will be determining how best to incorporate TRBC1. We present this comprehensive summary of TRBC1 and supplemental case examples to familiarize the flow cytometry community with its potential for routine application, provide examples of how to incorporate it into T-cell panels, and signal caution in interpreting the results in certain diagnostic scenarios where appropriate.
Asunto(s)
Citometría de Flujo , Linfocitos T , Citometría de Flujo/métodos , Citometría de Flujo/normas , Humanos , Linfocitos T/inmunología , Inmunofenotipificación/métodos , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/genéticaRESUMEN
The central role of target antigen density on chimeric antigen receptor T cell potency highlights the need for accurate measurement of antigen levels on clinical tumor samples. Here, we present a protocol for quantifying antigen density for six cell-surface antigens on neuroblastoma cells metastatic to bone marrow. We describe steps for patient sample acquisition, flow cytometry panel development, instrument setup, and compensation and detail procedures for running clinical samples and data analysis. For complete details on the use and execution of this protocol, please refer to Heitzeneder et al. (2022).1.
Asunto(s)
Neoplasias de la Médula Ósea , Neuroblastoma , Humanos , Médula Ósea , Citometría de Flujo , Inmunoterapia , Neuroblastoma/terapia , Neoplasias de la Médula Ósea/terapiaRESUMEN
Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.
Asunto(s)
Linfoma de Células B Grandes Difuso , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/terapia , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Inmunoterapia Adoptiva/métodos , Linfocitos T , Antígenos CD19/genética , Microambiente TumoralRESUMEN
Lymphocyte variant hypereosinophilic syndrome (LV-HES) is a rare cause of eosinophilia that is due to eosinophilipoietic cytokine production by an immunophenotypically abnormal T-cell clone. The molecular pathogenesis of this disorder is largely unknown and only 1 case of LV-HES with a pathogenic STAT3 mutation has been described thus far. Here we report 2 cases of LV-HES with STAT3 SH2 domain mutations. These cases further support the model that activation of STAT3 signaling through STAT3 SH2 domain mutations is a recurrent event in LV-HES.
Asunto(s)
Síndrome Hipereosinofílico/patología , Factor de Transcripción STAT3/genética , Linfocitos T/patología , Dominios Homologos src/genética , Adolescente , Adulto , Femenino , Humanos , Síndrome Hipereosinofílico/genética , Masculino , MutaciónRESUMEN
OBJECTIVES: Kikuchi-Fujimoto disease (KFD) and systemic lupus erythematosus (SLE) are benign entities with histologic features that raise concern about malignancy and infection. We searched for a histology-independent KFD/SLE signature relying on only immunophenotype and basic clinical characteristics. METHODS: A histology-independent KFD/SLE signature was generated using 975 excised lymph nodes with flow immunophenotyping, including 16 cases of KFD/SLE. This signature was then evaluated in 1,198 fine-needle aspiration (FNA) specimens. RESULTS: The top flow cytometry discriminant for KFD/SLE was uniform CD38+ expression on CD19+ events. Immunohistochemistry demonstrated nodules of IgD+, IgM- B cells surrounding necrotizing and activated T-cell areas. A signature combining 6 flow cytometry criteria with age and sample site had a positive predictive value of 88% for KFD/SLE, which had a prevalence of 1.6%. All 4 signature-positive FNA cases with follow-up excision were KFD/SLE. At a second institution, 4 of 5 KFD/SLE cases passed the top discriminant. CONCLUSIONS: A flow cytometry signature combined with age and biopsy site identifies KFD/SLE independent of histology, suggesting a shared immune composition and independently confirming that KFD/SLE represents a distinct entity. Unexpectedly, an IgD+CD38+ small B-cell population is a distinctive feature of KFD/SLE, suggesting a possible pathologic role for anergic/autoreactive B cells.
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Linfadenitis Necrotizante Histiocítica/diagnóstico , Lupus Eritematoso Sistémico/patología , Ganglios Linfáticos/patología , Linfadenitis/diagnóstico , Linfadenopatía/diagnóstico , Adolescente , Adulto , Niño , Preescolar , Femenino , Citometría de Flujo , Linfadenitis Necrotizante Histiocítica/patología , Humanos , Inmunofenotipificación , Linfadenitis/patología , Linfadenopatía/patología , MasculinoRESUMEN
The diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to 'see' like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of 'morphometric' markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases.
Asunto(s)
Leucemia/diagnóstico , Leucemia/patología , Linfoma/diagnóstico , Linfoma/patología , Análisis de la Célula Individual/métodos , Células Madre Hematopoyéticas/patología , Humanos , Laminas/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Células Mieloides/patología , Proteínas R-SNARE/metabolismoRESUMEN
Activation of the phosphoinositide 3-kinase (PI3K) pathway promotes proliferation and survival in many different cell types of the immune system. PI3K acts downstream of receptors that mediate proliferation and survival in T cells, and required roles for individual class I PI3K catalytic isoforms have been established. Interestingly, mice with either augmented or diminished PI3K activity in T cells develop lymphoproliferation and signs of autoimmunity. Here, we summarize our current knowledge of mouse strains with hyperactive or reduced PI3K, different isoforms of class I PI3K in T cell-mediated immunity and autoimmunity, and the therapeutic implications for modulating this pathway for treatment of various autoimmune diseases.
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Autoinmunidad , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Linfocitos T/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismoAsunto(s)
Mutación , Factores de Transcripción/genética , Humanos , Neoplasias , Proteínas Represoras/genéticaRESUMEN
Phosphoinositide kinase (PI3K) is activated by various receptors on lymphocytes and regulates development, activation, and tolerance. Genetic ablation of PI3K function in T cells leads to the appearance of autoimmune disorders. In B cells, loss of the class IA regulatory subunit p85alpha causes a partial defect in B cell development and proliferation, whereas loss of p85beta alone causes no apparent changes in B cell function. Here we investigate further the consequences of p85beta deletion in B cells, in the presence or absence of p85alpha. We demonstrate that p85beta partially compensates for loss of p85alpha in B cell development and peripheral survival, with greater defects observed when both isoforms are absent. BCR-mediated AKT phosphorylation is partially reduced in p85alpha-deficient B cells and further diminished with concomitant loss of p85beta. Unexpectedly, loss of p85beta results in increased BCR-mediated proliferation and ERK phosphorylation. These results indicate that the p85beta regulatory isoform has partially overlapping functions with p85alpha in B cells as well as a unique role in opposing BCR responses.
Asunto(s)
Linfocitos B/enzimología , Linfocitos B/inmunología , Isoenzimas , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Linfocitos B/citología , Diferenciación Celular , Isoenzimas/genética , Isoenzimas/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/genética , Transducción de SeñalRESUMEN
Ligation of the transmembrane protein T cell Ig and mucin domain (Tim)-1 can costimulate T cell activation. Agonistic Abs to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or costimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in a lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI3K. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Virales/metabolismo , Transducción de Señal/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD4-Positivos/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Interleucina-2/biosíntesis , Células Jurkat , Lectinas Tipo C , Glicoproteínas de Membrana/química , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Receptores Virales/química , Transfección , TirosinaRESUMEN
The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85alpha, p55alpha, p50alpha, and p85beta. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA-deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T/fisiología , Animales , Proliferación Celular , Técnicas In Vitro , Cooperación Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/clasificación , Fosfatidilinositol 3-Quinasas/deficiencia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Quinasas/metabolismo , Subunidades de Proteína , Transducción de Señal , Linfocitos T/citología , Linfocitos T/inmunología , Serina-Treonina Quinasas TORRESUMEN
Sjögren's syndrome (SS) is an autoimmune disease that is characterized by infiltration of exocrine tissues, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Here, we show that mice with T cell-specific loss of class IA phosphoinositide 3-kinase function develop organ-specific autoimmunity that resembles the human disease SS. Most mutant mice aged 3-8 months develop corneal opacity and eye lesions due to irritation and constant scratching. These mice display cardinal signs of primary SS such as marked lymphocytic infiltration of the lacrimal glands, antinuclear antibodies in the serum, and elevated titer of anti-SS-A antibody, in the absence of kidney pathology. Immunofluorescence studies show the presence of numerous CD4+ T cells with a smaller number of CD8+ T cells and B cells in the lacrimal glands. CD4+ T cells from these mice exhibit aberrant differentiation in vitro. These results indicate that aberrant T cells with impaired class IA phosphoinositide 3-kinase signaling can lead to organ-specific autoimmunity. In addition, the mouse model described here represents a tool to study the pathogenesis and treatment of SS.