RESUMO
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.
Assuntos
Antígenos CD19 , Imunoterapia Adotiva , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Linfoma Difuso de Grandes Células B/terapia , Linfoma Difuso de Grandes Células B/imunologia , Dose Máxima Tolerável , Receptores de Antígenos Quiméricos/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Idoso de 80 Anos ou maisRESUMO
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.
Assuntos
Citometria de Fluxo , Linfócitos T , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Linfócitos T/imunologia , Imunofenotipagem/métodos , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/genéticaRESUMO
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.
Assuntos
Neoplasias da Medula Óssea , Neuroblastoma , Humanos , Medula Óssea , Citometria de Fluxo , Imunoterapia , Neuroblastoma/terapia , Neoplasias da Medula Óssea/terapiaRESUMO
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.
Assuntos
Linfoma Difuso de Grandes Células B , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Linfoma Difuso de Grandes Células B/terapia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Imunoterapia Adotiva/métodos , Linfócitos T , Antígenos CD19/genética , Microambiente TumoralRESUMO
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.
Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Linfoma Difuso de Grandes Células B/terapia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Ensaios Clínicos Fase I como Assunto , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Prognóstico , Indução de RemissãoRESUMO
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.
Assuntos
Síndrome Hipereosinofílica/patologia , Fator de Transcrição STAT3/genética , Linfócitos T/patologia , Domínios de Homologia de src/genética , Adolescente , Adulto , Feminino , Humanos , Síndrome Hipereosinofílica/genética , Masculino , MutaçãoRESUMO
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.
Assuntos
Linfadenite Histiocítica Necrosante/diagnóstico , Lúpus Eritematoso Sistêmico/patologia , Linfonodos/patologia , Linfadenite/diagnóstico , Linfadenopatia/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Linfadenite Histiocítica Necrosante/patologia , Humanos , Imunofenotipagem , Linfadenite/patologia , Linfadenopatia/patologia , MasculinoRESUMO
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.
Assuntos
Leucemia/diagnóstico , Leucemia/patologia , Linfoma/diagnóstico , Linfoma/patologia , Análise de Célula Única/métodos , Células-Tronco Hematopoéticas/patologia , Humanos , Laminas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Células Mieloides/patologia , Proteínas R-SNARE/metabolismoRESUMO
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.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Linfócitos/citologia , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunossupressores/farmacologia , Linfócitos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Isoformas de Proteínas , Transdução de Sinais , Baço/citologia , Linfócitos T/citologia , Linfócitos T/enzimologiaRESUMO
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.
Assuntos
Linfócitos B/enzimologia , Linfócitos B/imunologia , Isoenzimas , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linfócitos B/citologia , Diferenciação Celular , Isoenzimas/genética , Isoenzimas/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/genética , Transdução de SinaisRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Virais/metabolismo , Transdução de Sinais/imunologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Interleucina-2/biossíntese , Células Jurkat , Lectinas Tipo C , Glicoproteínas de Membrana/química , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Receptores Virais/química , Transfecção , TirosinaRESUMO
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.
Assuntos
Autoimunidade , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Animais , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Linfócitos T/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
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.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Linfócitos T/fisiologia , Animais , Proliferação de Células , Técnicas In Vitro , Cooperação Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/classificação , Fosfatidilinositol 3-Quinases/deficiência , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases/metabolismo , Subunidades Proteicas , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Serina-Treonina Quinases TORRESUMO
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.