Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells.

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity. Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs). Results: Activated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature. Conclusion: These data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Pro-tumor Tfh2 cells induce detrimental IgG4 production and PGE2-dependent IgE inhibition in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and it is characterized by predominant pro-tumor Th2-type inflammation. T follicular helper (Tfh) cells are relevant immunoregulators in cancer, and often correlate with better survival. How the Th2-skewed microenvironment in PDAC modulates the differentiation of Tfh cells and their immunoregulatory function is unknown. METHODS: We carried out high-dimensional flow cytometry and T-cell receptor- and RNA-sequencing, as well as bioinformatics, immunohistochemistry and in vitro mechanistic studies. FINDINGS: We identified Tfh1-, Tfh2-, and Tfh17-like cell clusters in the blood, tumors and tumor-draining lymph-nodes (TDLNs) of chemo-naïve PDAC patients and showed that high percentages of Tfh2 cells within the tumor tissue and TDLNs correlated with reduced patient survival. Moreover, only Tfh2 cells were highly activated and were reduced in frequency in patients who responded to neoadjuvant chemotherapy. RNA-sequencing analysis of immunoglobulin expression showed that tumor and TDLN samples expressed all immunoglobulin (IGH) isotypes apart from IGHE. Consistent with these findings, Tfh2 cells differentiated in vitro by tumor microenvironment-conditioned dendritic cells promoted the production of anti-inflammatory IgG4 antibodies by co-cultured B cells, dependent on IL-13. Moreover, unexpectedly, Tfh2 cells inhibited the secretion of pro-inflammatory IgE, dependent on prostaglandin E2. INTERPRETATION: Our results indicate that in PDAC, highly activated pro-tumor Tfh2 favor anti-inflammatory IgG4 production, while inhibit pro-inflammatory IgE. Thus, targeting the circuits that drive Tfh2 cells, in combination with chemotherapy, may re-establish beneficial anti-tumor Tfh-B cell interactions and facilitate more effective treatment. FUNDING: Research grants from the Italian Association for Cancer Research (AIRC) IG-19119 to MPP and the AIRC Special Program in Metastatic disease: the key unmet need in oncology, 5 per Mille no. 22737 to CB, MF, CD, MR and MPP; the ERA-NET EuroNanoMed III (a collaborative european grant financed by the Italian Ministry of Health, Italy) project PANIPAC (JTC2018/041) to MPP; the Fondazione Valsecchi to SC.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Interferon regulatory factor 1 (IRF1) controls the metabolic programmes of low-grade pancreatic cancer cells.

OBJECTIVE: Pancreatic ductal adenocarcinomas (PDACs) include heterogeneous mixtures of low-grade cells forming pseudoglandular structures and compact nests of high-grade cells organised in non-glandular patterns. We previously reported that low-grade PDAC cells display high expression of interferon regulatory factor 1 (IRF1), a pivotal transcription factor of the interferon (IFN) system, suggesting grade-specific, cell-intrinsic activation of IFN responses. Here, we set out to determine the molecular bases and the functional impact of the activation of IFN-regulated responses in human PDACs. DESIGN: We first confirmed the correlation between glandular differentiation and molecular subtypes of PDAC on the one hand, and the expression of IRF1 and IFN-stimulated genes on the other. We next used unbiased omics approaches to systematically analyse basal and IFN-regulated responses in low-grade and high-grade PDAC cells, as well as the impact of IRF1 on gene expression programmes and metabolic profiles of PDAC cells. RESULTS: High-level expression of IRF1 in low-grade PDAC cells was controlled by endodermal lineage-determining transcription factors. IRF1-regulated gene expression equipped low-grade PDAC cells with distinctive properties related to antigen presentation and processing as well as responsiveness to IFN stimulation. Notably, IRF1 also controlled the characteristic metabolic profile of low-grade PDAC cells, suppressing both mitochondrial respiration and fatty acid synthesis, which may in part explain its growth-inhibiting activity. CONCLUSION: IRF1 links endodermal differentiation to the expression of genes controlling antigen presentation and processing as well as to the specification of the metabolic profile characteristic of classical PDAC cells.

Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis.

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma (PDAC). When occurring in the context of pancreatitis, KRAS mutations accelerate tumor development in mouse models. We report that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, pancreatic epithelial cells display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thereby limiting tissue damage through a rapid decrease of zymogen production. We propose that because activating mutations of KRAS maintain an irreversible ADM, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.

Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells.

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

Pancreatic Cancer Cells Require the Transcription Factor MYRF to Maintain ER Homeostasis.

Many tumors of endodermal origin are composed of highly secretory cancer cells that must adapt endoplasmic reticulum (ER) activity to enable proper folding of secreted proteins and prevent ER stress. We found that pancreatic ductal adenocarcinomas (PDACs) overexpress the myelin regulatory factor (MYRF), an ER membrane-associated transcription factor (TF) released by self-cleavage. MYRF was expressed in the well-differentiated secretory cancer cells, but not in the poorly differentiated quasi-mesenchymal cells that coexist in the same tumor. MYRF expression was controlled by the epithelial identity TF HNF1B, and it acted to fine-tune the expression of genes encoding highly glycosylated, cysteine-rich secretory proteins, thus preventing ER overload. MYRF-deficient PDAC cells showed signs of ER stress, impaired proliferation, and an inability to form spheroids in vitro, while in vivo they generated highly secretory but poorly proliferating and hypocellular tumors. These data indicate a role of MYRF in the control of ER homeostasis in highly secretory PDAC cells.

A molecular network regulating the proinflammatory phenotype of human memory T lymphocytes.

Understanding the mechanisms that modulate helper T lymphocyte functions is crucial to decipher normal and pathogenic immune responses in humans. To identify molecular determinants influencing the pathogenicity of T cells, we separated ex vivo-isolated primary human memory T lymphocytes on the basis of their ability to produce high levels of inflammatory cytokines. We found that the inflammatory, cytokine-producing phenotype of memory T lymphocytes was defined by a specific core gene signature and was mechanistically regulated by the constitutive activation of the NF-κB pathway and by the expression of the transcriptional repressor BHLHE40. BHLHE40 attenuated the expression of anti-inflammatory factors, including miR-146a, a negative regulator of NF-κB activation and ZC3H12D, an RNase of the Regnase-1 family able to degrade inflammatory transcripts. Our data reveal a molecular network regulating the proinflammatory phenotype of human memory T lymphocytes, with the potential to contribute to disease.

WDR5 inhibition halts metastasis dissemination by repressing the mesenchymal phenotype of breast cancer cells.

BACKGROUND: Development of metastases and drug resistance are still a challenge for a successful systemic treatment in breast cancer (BC) patients. One of the mechanisms that confer metastatic properties to the cell relies in the epithelial-to-mesenchymal transition (EMT). Moreover, both EMT and metastasis are partly modulated through epigenetic mechanisms, by repression or induction of specific related genes. METHODS: We applied shRNAs and drug targeting approaches in BC cell lines and metastatic patient-derived xenograft (PDX) models to inhibit WDR5, the core subunit of histone H3 K4 methyltransferase complexes, and evaluate its role in metastasis regulation. RESULT: We report that WDR5 is crucial in regulating tumorigenesis and metastasis spreading during BC progression. In particular, WDR5 loss reduces the metastatic properties of the cells by reverting the mesenchymal phenotype of triple negative- and luminal B-derived cells, thus inducing an epithelial trait. We also suggest that this regulation is mediated by TGFß1, implying a prominent role of WDR5 in driving EMT through TGFß1 activation. Moreover, such EMT reversion can be induced by drug targeting of WDR5 as well, leading to BC cell sensitization to chemotherapy and enhancement of paclitaxel-dependent effects. CONCLUSIONS: We suggest that WDR5 inhibition could be a promising pharmacologic approach to reduce cell migration, revert EMT, and block metastasis formation in BC, thus overcoming resistance to standard treatments.

FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner.

Differentiation of normal and tumor cells is controlled by regulatory networks enforced by lineage-determining transcription factors (TFs). Among them, TFs such as FOXA1/2 bind naïve chromatin and induce its accessibility, thus establishing new gene regulatory networks. Pancreatic ductal adenocarcinoma (PDAC) is characterized by the coexistence of well- and poorly differentiated cells at all stages of disease. How the transcriptional networks determining such massive cellular heterogeneity are established remains to be determined. We found that FOXA2, a TF controlling pancreas specification, broadly contributed to the cis-regulatory networks of PDACs. Despite being expressed in both well- and poorly differentiated PDAC cells, FOXA2 displayed extensively different genomic distributions and controlled distinct gene expression programs. Grade-specific functions of FOXA2 depended on its partnership with TFs whose expression varied depending on the differentiation grade. These data suggest that FOXA2 contributes to the regulatory networks of heterogeneous PDAC cells via interactions with alternative partner TFs.

Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity.

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

Dnmt3a restrains mast cell inflammatory responses.

DNA methylation and specifically the DNA methyltransferase enzyme DNMT3A are involved in the pathogenesis of a variety of hematological diseases and in regulating the function of immune cells. Although altered DNA methylation patterns and mutations in DNMT3A correlate with mast cell proliferative disorders in humans, the role of DNA methylation in mast cell biology is not understood. By using mast cells lacking Dnmt3a, we found that this enzyme is involved in restraining mast cell responses to acute and chronic stimuli, both in vitro and in vivo. The exacerbated mast cell responses observed in the absence of Dnmt3a were recapitulated or enhanced by treatment with the demethylating agent 5-aza-2'-deoxycytidine as well as by down-modulation of Dnmt1 expression, further supporting the role of DNA methylation in regulating mast cell activation. Mechanistically, these effects were in part mediated by the dysregulated expression of the scaffold protein IQGAP2, which is characterized by the ability to regulate a wide variety of biological processes. Altogether, our data demonstrate that DNMT3A and DNA methylation are key modulators of mast cell responsiveness to acute and chronic stimulation.

TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities.

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.

Dissection of transcriptional and cis-regulatory control of differentiation in human pancreatic cancer.

The histological grade of carcinomas describes the ability of tumor cells to organize in differentiated epithelial structures and has prognostic and therapeutic impact. Here, we show that differential usage of the genomic repertoire of transcriptional enhancers leads to grade-specific gene expression programs in human pancreatic ductal adenocarcinoma (PDAC). By integrating gene expression profiling, epigenomic footprinting, and loss-of-function experiments in PDAC cell lines of different grade, we identified the repertoires of enhancers specific to high- and low-grade PDACs and the cognate set of transcription factors acting to maintain their activity. Among the candidate regulators of PDAC differentiation, KLF5 was selectively expressed in pre-neoplastic lesions and low-grade primary PDACs and cell lines, where it maintained the acetylation of grade-specific enhancers, the expression of epithelial genes such as keratins and mucins, and the ability to organize glandular epithelia in xenografts. The identification of the transcription factors controlling differentiation in PDACs will help clarify the molecular bases of its heterogeneity and progression.

Energy metabolism characterization of a novel cancer stem cell-like line 3AB-OS.

Cancer stem cells (CSC) have a central role in driving tumor growth. Since metabolism is becoming an important diagnostic and therapeutic target, characterization of CSC line energetic properties is an emerging need. Embryonic and adult stem cells, compared to differentiated cells, exhibit a reduced mitochondrial activity and a stronger dependence on aerobic glycolysis. Here, we aimed to comparatively analyze bioenergetics features of the human osteosarcoma 3AB-OS CSC-like line, and the parental osteosarcoma MG63 cells, from which 3AB-OS cells have been previously selected. Our results suggest that 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, growth in glucose shortage or in presence of galactose or pyruvate (mitochondrial specific substrates) leads to a significant reduction of their proliferation compared to MG63 cells. Accordingly, 3AB-OS cells show an increased expression of lactate dehydrogenase A (LDHA) and a larger accumulation of lactate in the culture medium. In line with these findings 3AB-OS cells as compared to MG63 cells present a reduced mitochondrial respiration, a stronger sensitivity to glucose depletion or glycolysis inhibition and a lessened sensitivity to oxidative phosphorylation inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display fragmented mitochondria, which become networked as they grow in glucose-rich medium, while almost entirely loose these structures growing in low glucose. Overall, our findings suggest that 3AB-OS CSC energy metabolism is more similar to normal stem cells and to cancer cells characterized by a glycolytic anaerobic metabolism.

Overexpression of Far1, a cyclin-dependent kinase inhibitor, induces a large transcriptional reprogramming in which RNA synthesis senses Far1 in a Sfp1-mediated way.

The FAR1 gene encodes an 830 residue bifunctional protein, whose major function is inhibition of cyclin-dependent kinase complexes involved in the G1/S transition. FAR1 transcription is maximal between mitosis and early G1 phase. Enhanced FAR1 transcription is necessary but not sufficient for the pheromone-induced G1 arrest, since FAR1 overexpression itself does not trigger cell cycle arrest. Besides its well established role in the response to pheromone, recent evidences suggest that Far1 may also regulate the mitotic cell cycle progression: in particular, it has been proposed that Far1, together with the G1 cyclin Cln3, may be part of a cell sizer mechanism that controls the entry into S phase. Far1 is an unstable protein throughout the cell cycle except during G1 phase. Far1 levels peak in newborn cells as a consequence of a burst of synthetic activity at the end of the previous cycle, and the amounts per cell remain roughly constant during the G1 phase. Phosphorylation (at serine 87) by Cdk1-Cln complexes primes Far1 for ubiquitin-mediated proteolysis. By coupling a genome-wide transcriptional analysis of FAR1-overexpressing and far1Δ cells grown in ethanol- or glucose-supplemented minimal media with a range of phenotypic analysis, we show that FAR1 overexpression not only coordinately increases RNA and protein accumulation, but induces strong transcriptional remodeling, metabolism being the most affected cellular property, suggesting that the Far1/Cln3 sizer regulates cell growth either directly or indirectly by affecting metabolism and pathways known to modulate ribosome biogenesis. A crucial role in mediating the effect of Far1 overexpression is played by the Sfp1 protein, a key transcriptional regulator of ribosome biogenesis, whose presence is mandatory to allow a coordinated increase in both RNA and protein levels in ethanol-grown cells.

Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth.

Oncogenes such as K-ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of ¹5N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.