RESUMO
Targeted B-cell depletion is a useful therapy for many diseases, including autoimmune disorders and certain cancers. We developed a sensitive blood B-cell depletion assay, MRB 1.1, compared its performance with the T-cell/B-cell/NK-cell (TBNK) assay, and assessed B-cell depletion with different therapies. The empirically defined lower limit of quantification (LLOQ) for CD19+ cells in the TBNK assay was 10 cells/µL, and 0.441 cells/µL for the MRB 1.1 assay. The TBNK LLOQ was used to compare differences between B-cell depletion in similar lupus nephritis patient populations who received rituximab (LUNAR), ocrelizumab (BELONG), or obinutuzumab (NOBILITY). After 4 weeks, 10% of patients treated with rituximab retained detectable B cells vs 1.8% with ocrelizumab and 1.7% for obinutuzumab; at 24 weeks 93% of patients who received obinutuzumab remained below LLOQ vs 63% for rituximab. More-sensitive measurements of B cells may reveal differences in potency among anti-CD20 agents, which may associate with clinical outcomes.
Assuntos
Doenças Autoimunes , Linfócitos B , Humanos , Rituximab/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Células Matadoras NaturaisRESUMO
Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD28/metabolismo , Humanos , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antígeno B7-H1/genética , Antígeno B7-H1/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Plasmócitos/patologiaRESUMO
BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year survival rate for metastatic disease, yet with limited therapeutic advancements due to insufficient understanding of and inability to accurately capture high-risk CRC patients who are most likely to recur. We aimed to improve high-risk classification by identifying biological pathways associated with outcome in adjuvant stage II/III CRC. METHODS AND FINDINGS: We included 1062 patients with stage III or high-risk stage II colon carcinoma from the prospective three-arm randomized phase 3 AVANT trial, and performed expression profiling to identify a prognostic signature. Data from validation cohort GSE39582, The Cancer Genome Atlas, and cell lines were used to further validate the prognostic biology. Our retrospective analysis of the adjuvant AVANT trial uncovered a prognostic signature capturing three biological functions-stromal, proliferative and immune-that outperformed the Consensus Molecular Subtypes (CMS) and recurrence prediction signatures like Oncotype Dx in an independent cohort. Importantly, within the immune component, high granzyme B (GZMB) expression had a significant prognostic impact while other individual T-effector genes were less or not prognostic. In addition, we found GZMB to be endogenously expressed in CMS2 tumor cells and to be prognostic in a T cell independent fashion. A limitation of our study is that these results, although robust and derived from a large dataset, still need to be clinically validated in a prospective study. CONCLUSIONS: This work furthers our understanding of the underlying biology that propagates stage II/III CRC disease progression and provides scientific rationale for future high-risk stratification and targeted treatment evaluation in biomarker defined subpopulations of resectable high-risk CRC. Our results also shed light on an alternative GZMB source with context-specific implications on the disease's unique biology.
Assuntos
Neoplasias Colorretais/metabolismo , Granzimas/fisiologia , Transcriptoma , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Neoplasias Colorretais/mortalidade , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Granzimas/química , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Estudos Retrospectivos , Risco , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: CD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy. METHODS: Here, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)). RESULTS: ITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion. CONCLUSIONS: Our analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing antitumor T-cell response.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD/genética , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Cadeias alfa de Integrinas/genética , Neoplasias Pulmonares/tratamento farmacológico , Linfócitos do Interstício Tumoral/imunologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Anticorpos Monoclonais Humanizados/efeitos adversos , Antígeno B7-H1/imunologia , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Fenótipo , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Tempo , Resultado do Tratamento , Microambiente Tumoral , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/imunologiaRESUMO
Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.
Assuntos
Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/patologia , Variantes Farmacogenômicos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Células Clonais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linfócitos T/metabolismo , TranscriptomaRESUMO
Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm has emerged as a popular tool for visualizing high-parameter single-cell data. While this approach has obvious potential for data visualization it remains unclear how t-SNE analysis compares to conventional manual hand-gating in stratifying and quantitating the frequency of diverse immune cell populations. We applied a comprehensive 38-parameter mass cytometry panel to human blood and compared the frequencies of 28 immune cell subsets using both conventional bivariate and t-SNE-guided manual gating. t-SNE analysis was capable of stratifying every general cellular lineage and most sub-lineages with high correlation between conventional and t-SNE-guided cell frequency calculations. However, specific immune cell subsets delineated by the manual gating of continuous variables were not fully separated in t-SNE space thus causing discrepancies in subset identification and quantification between these analytical approaches. Overall, these studies highlight the consistency between t-SNE and conventional hand-gating in stratifying general immune cell lineages while demonstrating that particular cell subsets defined by conventional manual gating may be intermingled in t-SNE space.
Assuntos
Citometria de Fluxo/métodos , Análise de Célula Única/métodos , Linfócitos T/citologia , Linfócitos T/imunologia , Humanos , Análise de Componente PrincipalRESUMO
Signal interference or overlap in mass cytometry is minimal compared to flow cytometry but must still be considered for optimal panel design and assay sensitivity. Here we describe a procedure for evaluating signal interference dynamics in the context of a 25-parameter core immunophenotyping panel. Specifically, a mass-minus-many (MMM) approach was used to assess background signals in "empty" or "blank" channels intended for further customization. Through this approach cell type-specific variability in signal background is revealed. Further panel customization can thus be performed with an understanding of cell type and channel-specific background levels to enable rational panel design and the objective delineation of gating thresholds during analysis.
Assuntos
Biomarcadores/análise , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Leucócitos Mononucleares/citologia , Espectrometria de Massas/métodos , Análise de Célula Única/métodos , Humanos , Razão Sinal-RuídoRESUMO
Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti- integrin ß7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in ß7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. © 2016 International Society for Advancement of Cytometry.
Assuntos
Anticorpos Monoclonais/imunologia , Citometria de Fluxo/métodos , Cadeias beta de Integrinas/biossíntese , Leucócitos Mononucleares/metabolismo , Anticorpos Monoclonais/química , Regulação da Expressão Gênica , Humanos , Cadeias beta de Integrinas/imunologia , Leucócitos Mononucleares/ultraestruturaRESUMO
BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. METHODS: We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. RESULTS: The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. CONCLUSIONS: We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo/métodos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Biomarcadores Tumorais/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Indazóis/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Sulfonamidas/farmacologiaRESUMO
To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type beta transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.