RESUMO
The cellular source of positive signals that reinvigorate T cells within the tumor microenvironment (TME) for the therapeutic efficacy of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade has not been clearly defined. We now show that Batf3-lineage dendritic cells (DCs) are essential in this process. Flow cytometric analysis, gene-targeted mice, and blocking antibody studies revealed that 4-1BBL is a major positive co-stimulatory signal provided by these DCs within the TME that translates to CD8+ T cell functional reinvigoration and tumor regression. Immunofluorescence and spatial transcriptomics on human tumor samples revealed clustering of Batf3+ DCs and CD8+ T cells, which correlates with anti-PD-1 efficacy. In addition, proximity to Batf3+ DCs within the TME is associated with CD8+ T cell transcriptional states linked to anti-PD-1 response. Our results demonstrate that Batf3+ DCs within the TME are critical for PD-1/PD-L1 blockade efficacy and indicate a major role for the 4-1BB/4-1BB ligand (4-1BBL) axis during this process.
Assuntos
Antígeno B7-H1 , Fatores de Transcrição de Zíper de Leucina Básica , Linfócitos T CD8-Positivos , Células Dendríticas , Receptor de Morte Celular Programada 1 , Proteínas Repressoras , Microambiente Tumoral , Animais , Humanos , Camundongos , Ligante 4-1BB/metabolismo , Ligante 4-1BB/genética , Antígeno B7-H1/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
BACKGROUND: To describe an oncolytic adenovirus (OAd) encoding SP-SA-E7-4-1BBL that is capable of inducing tumor regression in therapeutic assays. Herein, we tested whether the antitumor effect is given by the induction of a tumor-specific immune response, as well as the minimum dose needed to elicit antitumor protection and monitor the OAd biodistribution over time. METHODS AND RESULTS: C57BL/6 mice (n = 5) per group were immunized twice with OAds encoding SP-SA-E7-4-1BBL, SA-E7-4-1BBL, or SP-SA-4-1BBL and challenged with TC-1 cancer cells. The DNA construct SP-SA-E7-4-1BBL was employed as a control via biolistic or PBS injection. Groups without tumor development at 47 days were rechallenged with TC-1 cells, and follow-up lasted until day 90. The minimum dose of OAd to induce the antitumor effect was established by immunization using serial dilution doses. The cytometry bead assay and the ELISpot assay were used to evaluate cytokine release in response to ex vivo antigenic stimulation. The distribution profile of the OAd vaccine was evaluated in the different organs by histological, immunohistochemical and qPCR analyses. The OAd SP-SA-E7-4-1BBL-immunized mice did not develop tumors even in a rechallenge. A protective antitumor effect was observed from a dose that is one hundredth of most reports of adenoviral vaccines. Immunization with OAd increases Interferon-gamma-producing cells in response to antigen stimulation. OAd was detected in tumors over time, with significant morphological changes, contrary to nontumor tissues. CONCLUSIONS: The OAd SP-SA-E7-4-1BBL vaccine confers a prophylactic, safe, long-lasting, and antigen-dependent antitumor effect mediated by a Th1 antitumor immune response.
Assuntos
Vacinas Anticâncer , Neoplasias , Animais , Camundongos , Papillomavirus Humano 16 , Ligante 4-1BB/genética , Ligante 4-1BB/farmacologia , Distribuição Tecidual , Camundongos Endogâmicos C57BL , Adenoviridae/genética , Imunidade , Neoplasias/terapiaRESUMO
ABSTRACT: Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.
Assuntos
Modelos Animais de Doenças , Fibrose , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Remodelação Ventricular , Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/fisiopatologia , Remodelação Ventricular/efeitos dos fármacos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Masculino , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Miofibroblastos/enzimologia , Sistema de Sinalização das MAP Quinases , Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/enzimologia , Ligante 4-1BB/metabolismo , Ligante 4-1BB/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Actinas/metabolismo , Células Cultivadas , Transdução de Sinais , Movimento Celular , Camundongos , Função Ventricular Esquerda , Diferenciação Celular , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/efeitos dos fármacosRESUMO
Targeting co-stimulatory receptors promotes the activation and effector functions of anti-tumor lymphocytes. 4-1BB (CD137/TNFSF9), a member of the tumor necrosis factor receptor superfamily (TNFR-SF), is a potent co-stimulatory receptor that plays a prominent role in augmenting effector functions of CD8+ T cells, but also CD4+ T cells and NK cells. Agonistic antibodies against 4-1BB have entered clinical trials and shown signs of therapeutic efficacy. Here, we have used a T cell reporter system to evaluate various formats of 4-1BBL regarding their capacity to functionally engage its receptor. We found that a secreted 4-1BBL ectodomain harboring a trimerization domain derived from human collagen (s4-1BBL-TriXVIII) is a strong inducer of 4-1BB co-stimulation. Similar to the 4-1BB agonistic antibody urelumab, s4-1BBL-TriXVIII is very potent in inducing CD8+ and CD4+ T cell proliferation. We provide first evidence that s4-1BBL-TriXVIII can be used as an effective immunomodulatory payload in therapeutic viral vectors. Oncolytic measles viruses encoding s4-1BBL-TriXVIII significantly reduced tumor burden in a CD34+ humanized mouse model, whereas measles viruses lacking s4-1BBL-TriXVIII were not effective. Natural soluble 4-1BB ligand harboring a trimerization domain might have utility in tumor therapy especially when delivered to tumor tissue as systemic administration might induce liver toxicity.
Assuntos
Ligante 4-1BB , Linfócitos T CD8-Positivos , Camundongos , Animais , Humanos , Ligante 4-1BB/genética , Agentes de Imunomodulação , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral , Células Matadoras NaturaisRESUMO
Renal carcinoma progresses aggressively in patients with metastatic disease while curative strategies are limited. Here, we constructed a recombinant non-replicating adenovirus (Ad) vaccine encoding an immune activator, CD137L, and a tumor antigen, CAIX, for treating renal carcinoma. In a subcutaneous tumor model, tumor growth was significantly suppressed in the Ad-CD137L/CAIX vaccine group compared with the single vaccine group. The induction and maturity of CD11C+ and CD8+CD11C+ dendritic cell (DC) subsets were promoted in Ad-CD137L/CAIX co-immunized mice. Furthermore, the Ad-CD137L/CAIX vaccine elicited stronger tumor-specific multifunctional CD8+ T cell immune responses as demonstrated by increased proliferation and cytolytic function of CD8+ T cells. Notably, depletion of CD8+ T cells greatly compromised the effective protection provided by Ad-CD137L/CAIX vaccine, suggesting an irreplaceable role of CD8+ T cells for the immunopotency of the vaccine. In both lung metastatic and orthotopic models, Ad-CD137L/CAIX vaccine treatment significantly decreased tumor metastasis and progression and increased the induction of tumor-specific multifunctional CD8+ T cells, in contrast to treatment with the Ad-CAIX vaccine alone. The Ad-CD137L/CAIX vaccine also augmented the tumor-specific multifunctional CD8+ T cell immune response in both orthotopic and metastatic models. These results indicated that Ad-CD137L/CAIX vaccine elicited a potent anti-tumor activity by inducing CD8+DC-mediated multifunctional CD8+ T cell immune responses. The potential strategy of CD137L-based vaccine might be served as a novel treatment for renal carcinoma or other malignant tumors.
Assuntos
Ligante 4-1BB/genética , Vacinas contra Adenovirus/uso terapêutico , Anidrase Carbônica IX/genética , Carcinoma de Células Renais/terapia , Neoplasias Renais/terapia , Neoplasias Pulmonares/terapia , Ligante 4-1BB/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Anidrase Carbônica IX/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Células Dendríticas/imunologia , Feminino , Células HEK293 , Humanos , Interleucina-6/sangue , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB CRESUMO
INTRODUCTION: Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), and provides a target for a dendritic cell (DC) vaccine. CD137 ligand (CD137L) expressed on antigen presenting cells, costimulates CD137-expressing T cells, and reverse CD137L signaling differentiates monocytes to CD137L-DC, a type of DC, which is more potent than classical DC in stimulating T cells. METHODS: In this phase I study, patients with locally recurrent or metastatic NPC were administered CD137L-DC pulsed with EBV antigens (CD137L-DC-EBV-VAX). RESULTS: Of the 12 patients treated, 9 received full 7 vaccine doses with a mean administered cell count of 23.9 × 106 per dose. Treatment was well tolerated with only 4 cases of grade 1 related adverse events. A partial response was obtained in 1 patient, and 4 patients are still benefitting from a progression free survival (PFS) of currently 2-3 years. The mean pre-treatment neutrophil: lymphocyte ratio was 3.4 and a value of less than 3 was associated with prolonged median PFS. Progressors were characterized by a high frequency of naïve T cells but a low frequency of CD8+ effector T cells while patients with a clinical benefit (CB) had a high frequency of memory T cells. Patients with CB had lower plasma EBV DNA levels, and a reduction after vaccination. CONCLUSION: CD137L-DC-EBV-VAX was well tolerated. The use of CD137L-DC-EBV-VAX is demonstrated to be safe. Consistent results were obtained from all 12 patients, indicating that CD137L-DC-EBV-VAX induces an anti-EBV and anti-NPC immune response, and warranting further studies in patients post effective chemotherapy. PRECIS: The first clinical testing of CD137L-DC, a new type of monocyte-derived DC, finds that CD137L-DC are safe, and that they can induce an immune response against Epstein-Barr virus-associated nasopharyngeal carcinoma that leads to tumor regression or prevents tumor progression.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Ligante 4-1BB/genética , Células Dendríticas , Herpesvirus Humano 4 , Humanos , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapiaRESUMO
Early metastasis of pancreatic cancer (PC) leads to high mortality, and the underlying mechanism of metastasis remains unclear. Tumor necrosis factor superfamily member 9 (TNFSF9) is associated with poor prognosis in PC. Here, we investigated the effect of TNFSF9 on PC proliferation and apoptosis, and focused on the effect of TNFSF9 on PC metastasis and its potential mechanism. We found that TNFSF9 promotes PC metastasis in vivo and in vitro, and may be partially dependent on the Wnt/Snail signaling pathway. In addition, TNFSF9 also regulates the release of cytokines IL-10 and transforming growth factor-ß (TGF-ß) in pancreatic cancer cells through Wnt signaling to induce the M2 polarization of macrophages and promote the migration of PC cells. Overall, our study found that TNFSF9 may directly promote PC metastasis or indirectly promote PC metastasis through macrophage M2 polarization. Our study provides a new costimulatory target for the treatment of PC.
Assuntos
Ligante 4-1BB/metabolismo , Proliferação de Células , Macrófagos/fisiologia , Neoplasias Pancreáticas/patologia , Ligante 4-1BB/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-10/metabolismo , Metástase Linfática , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.
Assuntos
Ligante 4-1BB/genética , Terapia Baseada em Transplante de Células e Tecidos , Eritrócitos/metabolismo , Expressão Gênica , Terapia Genética , Interleucina-15/genética , Ligante 4-1BB/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Precursoras Eritroides/metabolismo , Feminino , Citometria de Fluxo , Genes Reporter , Engenharia Genética , Terapia Genética/métodos , Humanos , Interleucina-15/metabolismo , Camundongos , Modelos Animais , Ligação Proteica , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential 'off-the-shelf' in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.
Assuntos
Células Apresentadoras de Antígenos/transplante , Engenharia Celular/métodos , Eritrócitos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Ligante 4-1BB/genética , Ligante 4-1BB/imunologia , Ligante 4-1BB/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Modelos Animais de Doenças , Eritrócitos/metabolismo , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Ativação Linfocitária , Neoplasias/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/metabolismo , Cultura Primária de Células , Linfócitos T/imunologia , Linfócitos T/transplante , Transplante Homólogo/métodosRESUMO
Rhabdomyosarcoma (RMS) is a heterogeneous soft tissue neoplasm most frequently found in children and adolescents. As the prognosis for recurrent and metastatic RMS remains poor, immunotherapies are hoped to improve quality of life and survival. CD137 is a member of tumor necrosis factor receptor family and a T cell costimulatory molecule which induces potent cellular immune responses that are able to eliminate malignant cells. Therefore, it was puzzling to find expression of CD137 on an RMS tissue microarray by multiplex staining. CD137 is not only expressed by infiltrating T cells but also by malignant RMS cells. Functional in vitro experiments demonstrate that CD137 on RMS cells is being transferred to adjacent antigen-presenting cells by trogocytosis, where it downregulates CD137 ligand, and thereby reduces T cell costimulation which results in reduced killing of RMS cells. The transfer of CD137 and the subsequent downregulation of CD137 ligand is a physiological negative feedback mechanism that is likely usurped by RMS, and may facilitate its escape from immune surveillance. In addition, CD137 signals into RMS cells and induces IL-6 and IL-8 secretion, which are linked to RMS metastasis and poor prognosis. However, the ectopic expression of CD137 on RMS cells is an Achilles' heel that may be utilized for immunotherapy. Natural killer cells expressing an anti-CD137 chimeric antigen receptor specifically kill CD137-expressing RMS cells. Our study implicates ectopic CD137 expression as a pathogenesis mechanism in RMS, and it demonstrates that CD137 may be a novel target for immunotherapy of RMS.
Assuntos
Qualidade de Vida , Rabdomiossarcoma , Ligante 4-1BB/genética , Adolescente , Criança , Humanos , Imunoterapia , Rabdomiossarcoma/genética , Linfócitos T , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
The prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic transplantation is dismal when treated with conventional approaches. While single-target CD19 or CD22 chimeric antigen receptor (CAR) T-cell therapy has achieved high complete remission (CR) rates in refractory/relapsed B-ALL, it could not maintain a durable remission in most patients. To prolong relapse-free survival, we sequentially combined CD19 and CD22 CAR-T cells to treat post-transplant relapsed B-ALL patients with both CD19/CD22 antigen expression on lymphoblasts. Patient-derived donor cells were collected to produce CAR-T cells that were transfected by lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB. The second T-cell infusion was scheduled at least 1 month, and usually within 6 months after the first CAR-T treatment. Twenty-seven adult and pediatric patients, including 11 (41%) with extramedullary diseases (EMD), received the first CD19 CAR-T and 23 (85%) achieved CR. Subsequently, 21 out of 27 patients received the second CD22 CAR-T and were followed-up for a median of 19.7 (range, 5.6-27.3) months; 14 cases remained in CR, seven relapsed and two of them died from disease progression; Kaplan-Meier survival analysis showed overall survival and event-free survival rates of 88.5% and 67.5%, respectively, at both 12 months and 18 months. CAR-T associated graft-versus-host disease (GVHD) occurred in 23% of patients, with 8% new-onset acute GVHD and 15% persistent or worsened pre-existing cGVHD before CAR-T. This combination strategy of sequential CD19 and CD22 CAR-T therapy significantly improved the long-term survival in B-ALL patients who relapsed after transplantation.
Assuntos
Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Terapia de Salvação , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Ligante 4-1BB/genética , Adolescente , Adulto , Aloenxertos , Complexo CD3/genética , Criança , Pré-Escolar , Terapia Combinada , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Intervalo Livre de Progressão , Recidiva , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. METHODS: To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. RESULTS: With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. CONCLUSIONS: Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.
Assuntos
Ligante 4-1BB/genética , Perfilação da Expressão Gênica , Imunoterapia Adotiva , Leucemia/terapia , Neoplasias Ovarianas/terapia , Receptores de Antígenos Quiméricos/genética , Análise de Célula Única , Linfócitos T/transplante , Transcriptoma , Ligante 4-1BB/metabolismo , Animais , Citotoxicidade Imunológica/genética , Feminino , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Imunofenotipagem , Células K562 , Leucemia/genética , Leucemia/imunologia , Leucemia/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Fenótipo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host's inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8+ T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment.Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy.Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8+ T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA.Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8+ T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory.
Assuntos
Ligante 4-1BB/genética , Antígenos de Neoplasias/genética , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vaccinia virus/fisiologia , Ligante 4-1BB/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Terapia Combinada , Sinergismo Farmacológico , Feminino , Memória Imunológica , Melanoma Experimental/imunologia , Camundongos , Resultado do Tratamento , Microambiente Tumoral , Vaccinia virus/genéticaRESUMO
Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNγ stimulation. BVax migrates to key secondary lymphoid organs and is proficient at antigen cross-presentation, which promotes both the survival and the functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunological memory that prevented the growth of new tumors upon subsequent reinjection in cured mice. GBM patient-derived BVax was successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.
Assuntos
Ligante 4-1BB/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Vacinas Anticâncer/imunologia , Glioblastoma/terapia , Interferon gama/imunologia , Neoplasias Experimentais/terapia , Ligante 4-1BB/genética , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígenos CD40/genética , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/patologia , Interferon gama/genética , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologiaRESUMO
The tumor microenvironment (TME) contains noncancerous cells such as various types of immune cells and fibroblasts. Cancer cells direct these stromal cells to create a microenvironment favorable for tumor growth and intercellular interactions have a critical role in this process. In established tumors, interactions between CD137 and its ligand (CD137L) contribute to tumor immune evasion and tumor growth. Therefore, it is important to identify cells expressing CD137 and CD137L within tumors. In this chapter, we will introduce a simple, powerful method of analyzing CD137- and CD137L-expressing tumor cells using Fluorescence-activated cell sorting.
Assuntos
Ligante 4-1BB/metabolismo , Neoplasias/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Ligante 4-1BB/genética , Biomarcadores , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Expressão Gênica , Humanos , Imunofenotipagem/métodos , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.
Assuntos
Ligante 4-1BB/administração & dosagem , Antígenos de Neoplasias/administração & dosagem , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/administração & dosagem , Ligante 4-1BB/genética , Ligante 4-1BB/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Fibronectinas/genética , Fibronectinas/imunologia , Humanos , Camundongos , Neoplasias/imunologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.
Assuntos
Ligante 4-1BB/imunologia , Vacinas Anticâncer/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Ligante OX40/imunologia , Ligante 4-1BB/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Vesículas Extracelulares/genética , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/ultraestrutura , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Técnicas In Vitro , Ativação Linfocitária , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Ligante OX40/genéticaRESUMO
Aim: CD19 chimeric antigen receptor (CAR) T cells have been approved by the US FDA for treatment of relapsed and refractory (R/R) B-cell malignancies. Patients & methods: This study investigated the safety and efficacy of autologous 4-1BB costimulatory domain-engineered CD19 CAR-T cells in R/R B-cell lymphoma. Results: After CD19 CAR-T-cell infusion, severe cytokine release syndrome occurred in 28.6% (4/14) of the patients. The overall response rate was 77% with complete remission observed in 6/14 patients at 3 months. A higher tumor burden and grade 3-4 of myelosuppression after chemotherapy were associated with severe cytokine-release syndrome. Notably, combining CD19 CAR-T cells and PD-1 blockade, but not CD19 CAR-T cells alone, reduced intracranial tumor burden in a patient with central invasion of lymphoma. Conclusion: CD19 CAR-T cells could effectively induce tumor remission and PD-1 blockade might improve the efficacy in Chinese patients with R/R B-cell lymphoma.
Assuntos
Síndrome da Liberação de Citocina/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Ligante 4-1BB/genética , Ligante 4-1BB/metabolismo , Adulto , Antígenos CD19/genética , Antígenos CD19/metabolismo , Terapia Combinada , Síndrome da Liberação de Citocina/etiologia , Humanos , Linfoma de Células B/epidemiologia , Linfoma de Células B/imunologia , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Recidiva , Indução de Remissão , Carga Tumoral , Adulto JovemRESUMO
CD137 modulates type 1 diabetes (T1D) progression in NOD mice. We previously showed that CD137 expression in CD4 T cells inhibits T1D, but its expression in CD8 T cells promotes disease development by intrinsically enhancing the accumulation of ß-cell-autoreactive CD8 T cells. CD137 is expressed on a subset of FOXP3+ regulatory CD4 T cells (Tregs), and CD137+ Tregs are the main source of soluble CD137. Soluble CD137 suppresses T cells in vitro by binding to the CD137 ligand (CD137L) upregulated on activated T cells. To further study how the opposing functions of CD137 are regulated, we successfully targeted Tnfsf9 (encoding CD137L) in NOD mice using the CRISPR/Cas9 system (designated NOD.Tnfsf9 -/-). Relative to wild-type NOD mice, T1D development in the NOD.Tnfsf9 -/- strain was significantly delayed, and mice developed less insulitis and had reduced frequencies of ß-cell-autoreactive CD8 T cells. Bone marrow chimera experiments showed that CD137L-deficient hematopoietic cells were able to confer T1D resistance. Adoptive T cell transfer experiments showed that CD137L deficiency on myeloid APCs was associated with T1D suppression. Conversely, lack of CD137L on T cells enhanced their diabetogenic activity. Furthermore, neither CD137 nor CD137L was required for the development and homeostasis of FOXP3+ Tregs. However, CD137 was critical for the in vivo T1D-suppressive activity of FOXP3+ Tregs, suggesting that the interaction between CD137 and CD137L regulates their function. Collectively, our results provide new insights into the complex roles of CD137-CD137L interaction in T1D.
Assuntos
Ligante 4-1BB/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Linfócitos T Reguladores/metabolismo , Ligante 4-1BB/genética , Animais , Antígenos CD4/metabolismo , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Humanos , Tolerância Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Quimeras de Transplante , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Background: The immune-inducing effect of the quorum sensing (QS) molecule autoinducer-2 (AI-2) on macrophages has not been previously comprehensively studied.Methods: We performed proteomic analysis on macrophages cocultured with purified Fusobacterium nucleatum (F. nucleatum) AI-2 and performed western blot analysis to verify the differential protein expression. We further used the Gene Expression Profiling Interactive Analysis and Tumor Immune Estimation Resource databases to analyze the expression of differentially expressed proteins in microbial-associated digestive tract tumors.Results: Based on proteomic analysis, we identified 46 upregulated proteins and 11 downregulated proteins. The upregulated proteins were mostly inflammatory factors such as tumor necrosis factor ligand superfamily member 9 (TNFSF9). These proteins have a range of biological functions associated with the regulation of inflammatory responses, apoptosis and tumorigenesis. TNFSF9 is highly expressed in pancreatic adenocarcinoma (PAAD) tissues and is associated with M1 polarization of macrophages.Conclusions: Our data indicated that F. nucleatum AI-2 induced inflammatory responses and activated multiple signaling pathways in macrophages. TNFSF9 is the most significantly differentially expressed protein induced by F. nucleatum AI-2 and is involved in regulating immune cell infiltration in PAAD. Thus, AI-2 may become a new focus for studying the relationship between bacteria and cancer.