RESUMO
The rapid development of immune checkpoint blockade (ICB) therapies has revolutionized the cancer treatment landscape and brightened the long-term forecast for many cancer patients. However, the specific genomic and proteomic changes in tumors treated with different ICB treatments have yet to be fully characterized. We treated four non-small-cell lung carcinoma (NSCLC) tumor digests ex vivo with the anti-PD-L1 antibody durvalumab (D) alone or in combination with the anti-CTLA-4 antibody tremelimumab (T) to explore changes in gene and protein expression associated with these ICB therapies. All four tumors showed a robust increase in interferon gamma (IFN-γ) production (100-300% higher than isotype control) in both D- and D + T-treated tumors. Three of the four tumors showed additional increases in IFN-γ production with D + T compared with D (40-70%). A substantial reduction in interleukin 10 (IL-10) was also found in three of the four tumors (reduced to 4-8%) in response to D and D + T. Conventional CD4 + /CD8 + populations and T cell activation markers increased after D and D + T treatment. D and D + T upregulated multiple IPA pathways involving T cell activation. D + T resulted in additional upregulation of Th1/Th2 pathways through a different set of genes, as well as greater reduction in genes involved in epithelial-mesenchymal transition (EMT), angiogenesis, and cancer stemness. Our results demonstrated that D + T augmented the effects of D in the microenvironment of this set of NSCLC tumors. The specific impact of D + T on the regulation of EMT, angiogenesis, and cancer stemness warrants further evaluation in a larger set of tumors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteômica , Microambiente TumoralRESUMO
Humanized mouse models are important tools in many areas of biological drug development including, within oncology research, the development of antagonistic antibodies that have the potential to block tumor growth by controlling vascularization and are key to the generation of in vivo proof-of-concept efficacy data. However, due to cross reactivity between human antibodies and mouse target such studies regularly require mouse models expressing only the human version of the target molecule. Such humanized knock-in/knock-out, KIKO, models are dependent upon the generation of homozygous mice expressing only the human molecule, compensating for loss of the mouse form. However, KIKO strategies can fail to generate homozygous mice, even though the human form is expressed and the endogenous mouse locus is correctly targeted. A typical strategy for generating KIKO mice is by ATG fusion where the human cDNA is inserted downstream of the endogenous mouse promoter elements. However, when adopting this strategy it is possible that the mouse promoter fails to express the human form in a manner compensating for loss of the mouse form or alternatively the human protein is incompatible in the context of the mouse pathway being investigated. So to understand more around the biology of KIKO models, and to overcome our failure with a number of ATG fusion strategies, we developed a range of humanized models focused on Delta-like 4 (Dll4), a target where we initially failed to generate a humanized model. By adopting a broader biologic strategy, we successfully generated a humanized DLL4 KIKO which led to a greater understanding of critical biological aspects for consideration when developing humanized models.
Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos Transgênicos/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/farmacologia , Proteínas de Ligação ao Cálcio , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos Knockout , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.
Assuntos
Células Supressoras Mieloides/imunologia , Neoplasias/imunologia , Viroses/imunologia , Animais , Linhagem Celular Tumoral , Doença Crônica , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/imunologia , Feminino , Humanos , Tolerância Imunológica/genética , Interferon gama/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/classificação , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Supressoras Mieloides/classificação , Células Supressoras Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Transcriptoma , Viroses/genética , Viroses/metabolismoRESUMO
Tissue residency is considered a defining feature of the innate lymphoid cell (ILC) populations located within mucosal and adipose tissues. ILCs are also present within all lymphoid tissues, but whether ILCs migrate between lymphoid and nonlymphoid sites and in what context is poorly understood. To determine whether migratory ILCs exist within peripheral lymph nodes (LNs), we labeled all cells within the brachial LN (bLN) of transgenic mice expressing a photoconvertible fluorescent protein by direct exposure to light. Tracking of cellular changes in the labeled LN revealed the gradual migration of new ILCs into the tissue, balanced by egress of ILCs dependent on sphingosine-1-phosphate receptors. Most of the migratory ILCs were ILC1s, entering LNs directly from the circulation in a CD62L- and CCR7-dependent manner and thus behaving like conventional natural killer (cNK) cells. Upon egress, both ILC1s and cNK cells were found to recirculate through peripheral LNs. A distinct population of migratory ILC2s were detected in the LN, but most of the ILC3s were tissue resident. Functionally, both migratory and resident ILC1s within LNs were able to rapidly produce IFN-γ to support the generation of robust TH1 T cell responses after immunization. Thus, migratory and resident ILC populations exist within peripheral LNs, with ILC1s, akin to cNK cells, able to traffic into these tissues where they can contribute to the initiation of adaptive immunity.
Assuntos
Movimento Celular/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Linfonodos/citologia , Células Th1/imunologia , Animais , Citometria de Fluxo , Interferon gama/metabolismo , Selectina L/metabolismo , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores CCR7/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , TranscriptomaRESUMO
Purpose: To identify a predictive biomarker for durvalumab, an anti-programmed death ligand 1 (PD-L1) mAb.Experimental Design: RNA sequencing of 97 advanced-stage non-small cell lung carcinoma (NSCLC) biopsies from a nonrandomized phase Ib/II clinical trial (1108/NCT01693562) were profiled to identify a predictive signature; 62 locally advanced or metastatic urothelial cancer tumors from the same study were profiled to confirm predictive utility of the signature. Thirty NSCLC patients provided pre- and posttreatment tumors for messenger RNA (mRNA) analysis. NSCLC with ≥25% tumor cells and urothelial cancer with ≥25% tumor or immune cells stained for PD-L1 at any intensity were scored PD-L1 positive (PD-L1+). Kaplan-Meier and Cox proportional hazards analyses were used to adjust for gender, age, prior therapies, histology, ECOG status, liver metastasis, and smoking. Tumor mutation burden (TMB) was calculated using data from The Cancer Genome Atlas (TCGA).Results: In the NSCLC discovery set, a four-gene IFNγ-positive (IFNγ+) signature comprising IFNγ, CD274, LAG3, and CXCL9 was associated with higher overall response rates, longer median progression-free survival, and overall survival compared with signature-low patients. IFNγ+-signature NSCLC patients had improved survival regardless of IHC PD-L1 status. These associations were replicated in a urothelial cancer cohort. The IFNγ+ signature was induced 2-fold (P = 0.003) by durvalumab after 8 weeks of therapy in patients with NSCLC, and baseline signature was associated with TMB but not survival in TCGA data.Conclusions: The IFNγ+ mRNA signature may assist in identifying patients with improved outcomes with durvalumab, independent of PD-L1 assessed by IHC. Clin Cancer Res; 24(16); 3857-66. ©2018 AACR.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígeno B7-H1/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Interferon gama/genética , Neoplasias Urológicas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Biópsia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimiocina CXCL9/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , RNA Mensageiro/genética , Resultado do Tratamento , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Urotélio/patologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
OBJECTIVE: B cells impact the progression of systemic sclerosis (SSc; scleroderma) through multiple pathogenic mechanisms. CD19 inhibition in mice reduced skin thickness, collagen production, and autoantibody levels, consistent with CD19 expression on plasma cells (PCs), the source of antibody production. PC depletion could effectively reduce collagen deposition and inflammation in SSc; therefore, we investigated the effects of PC depletion on SSc disease activity. METHODS: A PC gene signature was evaluated in SSc skin biopsy samples in 2 phase I clinical trials. We assessed microarray data from tissue from public studies of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), dermatomyositis (DM), systemic lupus erythematosus (SLE), and atopic dermatitis, as well as blood from a phase IIb clinical trial in SLE. RESULTS: The PC signature was elevated in SSc skin specimens compared to healthy donor skin (P = 2.28 × 10-6 ) and correlated with the baseline modified Rodnan skin thickness score (MRSS) (r = 0.64, P = 0.0004). Patients with a high PC signature at baseline showed greater improvement in the MRSS (mean ± SD change 35 ± 16%; P = 6.30 × 10-4 ) following anti-CD19 treatment with inebilizumab (MEDI-551) than did patients with a low PC signature at baseline (mean ± SD change 8 ± 12%; P = 0.104). The PC signature was overexpressed in tissue from patients with SLE, DM, COPD, interstitial lung disease, and IPF relative to controls (all fold change >2; P < 0.001). The PC signature also differed significantly between SLE patients with mild-to-moderate disease and those with severe disease (SLE Disease Activity Index cutoff at 10) (fold change 1.44; P = 3.90 × 10-3 ) and correlated significantly with the degree of emphysema in COPD (r = 0.53, P = 7.55 × 10-8 ). CONCLUSION: Our results support the notion that PCs have a role in the pathogenesis of SSc and other autoimmune or pulmonary indications. An elevated pretreatment PC signature was associated with increased benefit from MEDI-551 in SSc.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Plasmócitos/metabolismo , Escleroderma Sistêmico/tratamento farmacológico , Índice de Gravidade de Doença , Adulto , Biópsia , Método Duplo-Cego , Feminino , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/patologia , Masculino , Plasmócitos/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/patologia , Escleroderma Sistêmico/patologia , Pele/patologia , Resultado do TratamentoRESUMO
Lung fibrosis is an unabated wound healing response characterized by the loss and aberrant function of lung epithelial cells. Herein, we report that extracellular Clusterin promoted epithelial cell apoptosis whereas intracellular Clusterin maintained epithelium viability during lung repair. Unlike normal and COPD lungs, IPF lungs were characterized by significantly increased extracellular Clusterin whereas the inverse was evident for intracellular Clusterin. In vitro and in vivo studies demonstrated that extracellular Clusterin promoted epithelial cell apoptosis while intercellular Clusterin modulated the expression of the DNA repair proteins, MSH2, MSH6, OGG1 and BRCA1. The fibrotic response in Clusterin deficient (CLU-/-) mice persisted after bleomycin and it was associated with increased DNA damage, reduced DNA repair responses, and elevated cellular senescence. Remarkably, this pattern mirrored that observed in IPF lung tissues. Together, our results show that cellular localization of Clusterin leads to divergent effects on epithelial cell regeneration and lung repair during fibrosis.
Assuntos
Clusterina/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Idoso , Animais , Apoptose , Bleomicina/efeitos adversos , Estudos de Casos e Controles , Linhagem Celular , Clusterina/sangue , Clusterina/genética , Citoplasma/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo de Erro de Pareamento de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Células Epiteliais/patologia , Espaço Extracelular/metabolismo , Feminino , Fibrose , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/sangue , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologiaRESUMO
The heterogeneous clinical phenotypes of chronic obstructive pulmonary disease (COPD) challenge successful drug development. To identify COPD subgroups beyond clinical phenotypes, we interrogated blood immune cell profiles and ex-vivo responses of current and former smokers, with or without COPD, in the longitudinal COPD Genetic Epidemiology study (COPDGene) cohort. CD4+ and CD8+ T cells and monocytes were profiled by flow cytometry. Microarray analysis was performed on the RNA from the aforementioned isolated cells. T-cell directed whole blood ex-vivo stimulation was used to assess functional responses. Blood CD4+ T-cell transcript analysis distinguished patients with COPD from control smokers and also enriched for a subset of patients with COPD that had a history of exacerbations of the disease. Analogous analyses of CD8+ T cells and monocytes failed to discriminate patients with COPD from the control population. Patients with COPD had a diminished cytokine response, compared to control smokers, characterized by low levels of granulocyte-monocyte colony stimulation factor (GM-CSF), interferon gamma (IFN-É£), interleukin one-alpha (IL-1α), tumor necrosis factor-alpha (TNF-α) and tumor necrosis factor-beta (TNF-ß) secreted in response to T-cell directed ex-vivo stimulation. This cytokine response associated with baseline disease severity (forced expiratory volume in 1 second [FEV1]% predicted), rapidly declining lung function, and emphysema. Our observations indicate that COPD phenotypes can be further differentiated based on blood CD4+ T-cell profiles and resultant immune responses, suggesting a role for these cells in COPD pathophysiology.
RESUMO
OBJECTIVE: Production of pathogenic autoantibodies by self-reactive plasma cells (PCs) is a hallmark of autoimmune diseases. We undertook this study to investigate the prevalence of PCs and their relationship to known pathogenic pathways to increase our understanding of the role of PCs in disease progression and treatment response. METHODS: We developed a sensitive gene expression-based method to overcome the challenges of measuring PCs using flow cytometry. Whole-genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA1, IGJ, IGKC, IGKV4-1, and TNFRSF17, expressed predominantly in PCs. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy, for evaluating the relationship between PCs and other autoimmune disease-related genes, and for estimating PC levels in affected blood and tissue from patients with multiple autoimmune diseases. RESULTS: The PC signature was highly sensitive and capable of detecting a change in as few as 360 PCs. The PC signature was reduced more than 90% in scleroderma patients following anti-CD19 treatment, and this reduction was highly correlated (r = 0.80) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed that 30-35% of lupus and rheumatoid arthritis patients had increased levels of PCs. CONCLUSION: This newly developed PC signature provides a robust and accurate method of measuring PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases who may benefit from PC-depleting therapy.