RESUMEN
Costimulation blockade (CoB)-based immunotherapy is a promising alternative to immunosuppression for transplant recipients; however, the current limited understanding of the factors that impact its efficacy restrains its clinical applicability. In this context, pro- and anti-inflammatory cytokines are being recognized as having an impact on T cell activation beyond effector differentiation. This study aims at elucidating the impact of direct IL-10 signaling in T cells on CoB outcomes. We used a full-mismatch skin transplantation model where recipients had a T cell-restricted expression of a dominant negative IL-10 receptor (10R-DN), alongside anti-CD154 as CoB therapy. Unlike wild-type recipients, 10R-DN mice failed to benefit from CoB. This accelerated graft rejection correlated with increased accumulation of T cells producing TNF-α, IFN-γ, and IL-17. In vitro experiments indicated that while lack of IL-10 signaling did not change the ability of anti-CD154 to modulate alloreactive T cell proliferation, the absence of this pathway heightened TH1 effector cell differentiation. Furthermore, deficiency of IL-10 signaling in T cells impaired Treg induction, a hallmark of anti-CD154 therapy. Overall, these findings unveil an important and novel role of IL-10 signaling in T cells that defines the success of CoB therapies and identifies a target pathway for obtaining robust immunoregulation.
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.
Asunto(s)
Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Técnicas de Cocultivo , Transición Epitelial-Mesenquimal , Inflamación , Integrina beta1 , Neoplasias Pancreáticas , Análisis de la Célula Individual , Microambiente Tumoral , Humanos , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Inflamación/patología , Inflamación/metabolismo , Integrina beta1/metabolismo , Integrina beta1/genética , Organoides/patología , Organoides/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Comunicación CelularRESUMEN
PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.
Asunto(s)
Complejo CD3 , Endopeptidasas , Proteínas Ligadas a GPI , Inmunoterapia Adoptiva , Mesotelina , Neoplasias Pancreáticas , Receptores Quiméricos de Antígenos , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/inmunología , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Línea Celular Tumoral , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Adenocarcinoma/patologíaRESUMEN
Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging. SIGNIFICANCE: Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression. See related commentary by Isaacson et al., p. 1185.
Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias Pancreáticas , Animales , Ratones , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/uso terapéutico , Proteínas Proto-Oncogénicas c-akt , Neoplasias Pancreáticas/patología , Páncreas/patología , Fibroblastos/patología , Microambiente Tumoral , Línea Celular Tumoral , Fibroblastos Asociados al Cáncer/patologíaRESUMEN
BACKGROUND & AIMS: Myeloid cells are key regulators of cirrhosis, a major cause of mortality worldwide. Because stromal cells can modulate the functionality of myeloid cells in vitro, targeting stromal-myeloid interactions has become an attractive potential therapeutic strategy. We aimed to investigate how human liver stromal cells impact myeloid cell properties and to understand the utility of a stromal-myeloid coculture system to study these interactions in the context of cirrhosis. METHODS: Single-cell RNA-sequencing analyses of non-cirrhotic (n = 7) and cirrhotic (n = 5) human liver tissue were correlated to the bulk RNA-sequencing results of in vitro cocultured human CD14+ and primary liver stromal cells. Complimentary mechanistic experiments and flow cytometric analysis were performed on human liver stromal-myeloid coculture systems. RESULTS: We found that stromal-myeloid coculture reduces the frequency CD14+ cell subsets transcriptionally similar to liver macrophages, showing that stromal cells inhibit the maturation of monocytes into macrophages. Stromal cells also influenced in vitro macrophage differentiation by skewing away from cirrhosis-linked CD9+ scar-associated macrophage-like cells and towards CD163+ Kupffer cell-like macrophages. We identify IL-6 production as a mechanism by which stromal cells limit CD9+ macrophage differentiation and find that local IL-6 levels are decreased in early-stage human liver disease compared to healthy liver tissue, suggesting a protective role for local IL-6 in the healthy liver. CONCLUSIONS: Our work reveals an unanticipated role for liver stromal cells in impeding the maturation and altering the differentiation of macrophages and should prompt investigations into the role of local IL-6 production in the pathogenesis of liver disease. These studies provide a framework for investigating macrophage-stromal interactions during cirrhosis. LAY SUMMARY: The impact of human liver stromal cells on myeloid cell maturation and differentiation in liver disease is incompletely understood. In this study, we present a mechanistic analysis using a primary in vitro human liver stromal-myeloid coculture system that is translated to liver disease using single-cell RNA sequencing analysis of cirrhotic and non-cirrhotic human liver tissue. Our work supports a role for stromal cell contact in restricting macrophage maturation and for stromal-derived IL-6 in limiting the differentiation of a cirrhotic macrophage subset.
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Interleucina-6 , Hepatopatías , Diferenciación Celular , Humanos , Cirrosis Hepática/etiología , Hepatopatías/patología , Macrófagos/patología , Monocitos/patología , ARN , Células del Estroma/patologíaRESUMEN
Tumor involvement of major vascular structures limits surgical options in pancreatic adenocarcinoma (PDAC), which in turn limits opportunities for cure. Despite advances in locoregional approaches, there is currently no role for incomplete resection. This study evaluated a gelatinized neoantigen-targeted vaccine applied to a grossly positive resection margin in preventing local recurrence. Incomplete surgical resection was performed in mice bearing syngeneic flank Panc02 tumors, leaving a 1 mm rim adherent to the muscle bed. A previously validated vaccine consisting of neoantigen peptides, a stimulator of interferon genes (STING) agonist and AddaVaxTM (termed PancVax) was embedded in a hyaluronic acid hydrogel and applied to the tumor bed. Tumor remnants, regional lymph nodes, and spleens were analyzed using histology, flow cytometry, gene expression profiling, and ELISPOT assays. The immune microenvironment at the tumor margin after surgery alone was characterized by a transient influx of myeloid-derived suppressor cells (MDSCs), prolonged neutrophil influx, and near complete loss of cytotoxic T cells. Application of PancVax gel was associated with enhanced T cell activation in the draining lymph node and expansion of neoantigen-specific T cells in the spleen. Mice implanted with PancVax gel demonstrated no evidence of residual tumor at two weeks postoperatively and healed incisions at two months postoperatively without local recurrence. In summary, application of PancVax gel at a grossly positive tumor margin led to systemic expansion of neoantigen-specific T cells and effectively prevented local recurrence. These findings support further work into locoregional adjuncts to immune modulation in PDAC.
Asunto(s)
Adenocarcinoma , Vacunas contra el Cáncer , Neoplasias Pancreáticas , Adenocarcinoma/prevención & control , Adenocarcinoma/cirugía , Animales , Hidrogeles , Inmunoterapia , Ratones , Microambiente TumoralRESUMEN
The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA and TCR sequencing to detect and characterize "tumor-matching" (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared with nonmatching T cells in blood and were less exhausted than matching cells in tumors. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome, we identified candidate cell surface markers for TM cells in mice and patients and validated NKG2D, CD39, and CX3CR1 in mice. These data show that the TCR can be used to identify tumor-relevant cells for characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.