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PD-1 is a critical therapeutic target in cancer immunotherapy and antibodies blocking PD-1 are approved for multiple types of malignancies. The phosphatase SHP2 is the main effector mediating PD-1 downstream signaling and accordingly attempts have been made to target this enzyme as an alternative approach to treat immunogenic tumors. Unfortunately, small molecule inhibitors of SHP2 do not work as expected, suggesting that the role of SHP2 in T cells is more complex than initially hypothesized. To better understand the perplexing role of SHP2 in T cells, we performed interactome mapping of SAP, an adapter protein that is associated with SHP2 downstream signaling. Using genetic and pharmacological approaches, we discovered that SHP2 dephosphorylates ITK specifically downstream of PD-1 and that this event was associated with PD-1 inhibitory cellular functions. This study suggests that ITK is a unique target in this pathway, and since ITK is a SHP2-dependent specific mediator of PD-1 signaling, the combination of ITK inhibitors with PD-1 blockade may improve upon PD-1 monotherapy in the treatment of cancer.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.

PD-1 is an inhibitory receptor in T cells, and antibodies that block its interaction with ligands augment anti-tumor immune responses. The clinical potential of these agents is limited by the fact that half of all patients develop immune-related adverse events (irAEs). To generate insights into the cellular changes that occur during anti-PD-1 treatment, we performed single-cell RNA sequencing of circulating T cells collected from patients with cancer. Using the K-nearest-neighbor-based network graph-drawing layout, we show the involvement of distinctive genes and subpopulations of T cells. We identify that at baseline, patients with arthritis have fewer CD8 T

Ligation of the inhibitory receptor PD-1 on T cells results in the inhibition of numerous cellular functions. Despite the overtly inhibitory outcome of PD-1 signaling, an additional series of functions is activated. We have observed that T cells stimulated through the T cell receptor (TCR) and PD-1 primarily do not proliferate, however, there is a population of cells that proliferates more than through TCR stimulation alone. In this study, we performed flow cytometry analysis and RNA sequencing on individual populations of T cells and discovered that unlike the naive T cells, which were inhibited following PD-1 ligation, the T cells that proliferated more following PD-1 ligation were associated with the effector memory (EM) and central memory (CM) phenotypes. Remarkably, we showed that naive, EM, and CM T cell subsets had distinct gene expression profiles following PD-1 ligation. While in the naive and CM T cells, PD-1-regulated-genes were associated with T cell differentiation and metabolism, in the EM cells the genes regulated by PD-1 were coupled to more specific effector functions. Transcription factor analysis identified STAT1 and STAT2 as key mediators of PD-1-induced gene expression, and patients with low STAT1 and STAT2 expression responded favorably to check point blockade. Altogether, the presence of transcriptionally and functionally distinct T cell populations responsive to PD-1 ligation provide novel insights into the biology of PD-1 and suggest the use of T cell subset-specific approaches to improve the clinical outcome observed following therapeutic PD-1 blockade.

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions.

Checkpoints are inhibitory receptors expressed on activated T cells. During the process of carcinogenesis, tumor cells progressively express multiple inhibitory receptor-ligands to prevent T cell recognition and elimination. Consequently, therapeutic blockade of these checkpoints, or their ligands, helps recover anti-tumor immunity. Checkpoint inhibitors have been considered as a new armory for cancer patients due to the broad effectiveness of three agents blocking the inhibitory receptors CTLA-4, PD-1, and PD-L1 (the ligand for PD-1) (1). Based upon prolonged overall survival in clinical trials, antibodies inhibiting CTLA-4, PD-1 and PD-L1 have been approved by the FDA for multiple clinical indications, including melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, urothelial carcinoma, renal cell carcinoma, Hodgkin lymphoma, and other adult and pediatric solid tumors.

Summary Phospholipase C epsilon 1 (PLCε1) is a unique member of the phospholipase family, in that it also functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1. It is this function as a Rap1 GEF that gives PLCε1 an essential role in chemokine-mediated T cell adhesion. We have utilized a syngeneic tumor model, MC38 cells in C57BL/6 mice, and observed that tumors grow larger and more quickly in the absence of PLCε1. Single-cell analysis revealed an increased CD4+/CD8+ ratio in the spleens, lymph nodes and tumors of PLCε1 knock-out tumor-bearing mice. T cells isolated from PLCε1 knock-out mice were less activated by multiple phenotypical parameters than those from wild-type mice. We additionally noted a decrease in expression of the chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C motif chemokine receptor 4 (CCR4) on CD4+ T cells from the spleens, lymph nodes and tumors of PLCε1 knock-out mice compared to wild-type mice, and diminished migration of PLCε1-depleted CD3+ T cells towards stromal cell-derived factor (SDF)-1α. Based on these results, we conclude that PLCε1 is a potential regulator of tumor-infiltrating lymphocytes, functioning, at least in part, at the level of T cell trafficking and recruitment.

The tumor microenvironment (TME) plays key roles in promoting disease progression in the aggressive triple-negative subtype of breast cancer (TNBC; Basal/Basal-like). Here, we took an integrative approach and determined the impact of tumor-stroma-inflammation networks on pro-metastatic phenotypes in TNBC. With the TCGA dataset we found that the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β), as well as their target pro-metastatic chemokines CXCL8 (IL-8), CCL2 (MCP-1), and CCL5 (RANTES) were expressed at significantly higher levels in basal patients than luminal-A patients. Then, we found that TNFα- or IL-1β-stimulated co-cultures of TNBC cells (MDA-MB-231, MDA-MB-468, BT-549) with mesenchymal stem cells (MSCs) expressed significantly higher levels of CXCL8 compared to non-stimulated co-cultures or each cell type alone, with or without cytokine stimulation. CXCL8 was also up-regulated in TNBC co-cultures with breast cancer-associated fibroblasts (CAFs) derived from patients. CCL2 and CCL5 also reached the highest expression levels in TNFα/IL-1β-stimulated TNBC:MSC/CAF co-cultures. The elevations in CXCL8 and CCL2 expression partly depended on direct physical contacts between the tumor cells and the MSCs/CAFs, whereas CCL5 up-regulation was entirely dependent on cell-to-cell contacts. Supernatants of TNFα-stimulated TNBC:MSC “Contact” co-cultures induced robust endothelial cell migration and sprouting. TNBC cells co-cultured with MSCs and TNFα gained migration-related morphology and potent migratory properties; they also became more invasive when co-cultured with MSCs/CAFs in the presence of TNFα. Using siRNA to CXCL8, we found that CXCL8 was significantly involved in mediating the pro-metastatic activities gained by TNFα-stimulated TNBC:MSC “Contact” co-cultures: angiogenesis, migration-related morphology of the tumor cells, as well as cancer cell migration and invasion. Importantly, TNFα stimulation of TNBC:MSC “Contact” co-cultures in vitro has increased the aggressiveness of the tumor cells in vivo, leading to higher incidence of mice with lung metastases than non-stimulated TNBC:MSC co-cultures. Similar tumor-stromal-inflammation networks established in-culture with luminal-A cells demonstrated less effective or differently-active pro-metastatic functions than those of TNBC cells. Overall, our studies identify novel tumor-stroma-inflammation networks that may promote TNBC aggressiveness by increasing the pro-malignancy potential of the TME and of the tumor cells themselves, and reveal key roles for CXCL8 in mediating these metastasis-promoting activities.

Stromal cells and pro-inflammatory cytokines play key roles in promoting the aggressiveness of triple-negative breast cancers (TNBC; Basal/Basal-like). In our previous study we demonstrated that stimulation of TNBC and mesenchymal stem cells (MSCs) co-cultures by the pro-inflammatory cytokine tumor necrosis factor α (TNFα) has led to increased metastasis-related properties in vitro and in vivo. In this context, elevated release of the pro-metastatic chemokines CXCL8 (IL-8) and CCL5 (RANTES) was noted in TNFα- and interleukin-1β (IL-1β)-stimulated TNBC:MSC co-cultures; the process was partly (CXCL8) and entirely (CCL5) dependent on physical contacts between the two cell types. Here, we demonstrate that DAPT, inhibitor of γ-secretase that participates in activation of Notch receptors, inhibited the migration and invasion of TNBC cells that were grown in "Contact" co-cultures with MSCs or with patient-derived cancer-associated fibroblasts (CAFs), in the presence of TNFα. DAPT also inhibited the contact-dependent induction of CXCL8, but not of CCL5, in TNFα- and IL-1β-stimulated TNBC:MSC/CAF co-cultures; some level of heterogeneity between the responses of different TNBC cell lines was noted, with MDA-MB-231:MSC/CAF co-cultures being the most sensitive to DAPT. Patient dataset studies comparing basal tumors to luminal-A tumors, and mRNA analyses of Notch receptors in TNBC and luminal-A cells pointed at Notch1 as possible mediator of CXCL8 increase in TNFα-stimulated TNBC:stroma "Contact" co-cultures. Accordingly, down-regulation of Notch1 in TNBC cells by siRNA has substantially reduced the contact-dependent elevation in CXCL8 in TNFα- and also in IL-1β-stimulated TNBC:MSC "Contact" co-cultures. Then, studies in which CXCL8 or p65 (NF-κB pathway) were down-regulated (siRNAs; CRISPR/Cas9) in TNBC cells and/or MSCs, indicated that upon TNFα stimulation of "Contact" co-cultures, p65 was activated and led to CXCL8 production mainly in TNBC cells. Moreover, our findings indicated that when tumor cells interacted with stromal cells in the presence of pro-inflammatory stimuli, TNFα-induced p65 activation has led to elevated Notch1 expression and activation, which then gave rise to elevated production of CXCL8. Overall, tumor:stroma interactions set the stage for Notch1 activation by pro-inflammatory signals, leading to CXCL8 induction and consequently to pro-metastatic activities. These observations may have important clinical implications in designing novel therapy combinations in TNBC.

// Alexander Bott 1 , Nese Erdem 1 , Shalom Lerrer 2 , Agnes Hotz-Wagenblatt 3 , Christian Breunig 1 , Khalid Abnaof 1 , Angelika Worner 1 , Heike Wilhelm 1 , Ewald Munstermann 1 , Adit Ben-Baruch 2 and Stefan Wiemann 1 1 Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel 3 Bioinformatics Group, Genomics & Proteomics Core Facility (GPCF), German Cancer Research Center (DKFZ), Heidelberg, Germany Correspondence to: Alexander Bott, email: alex.bott@gmx.de Stefan Wiemann, email: s.wiemann@dkfz.de Keywords: breast cancer, tumor microenvironment, mesenchymal stem/stromal cell, microRNA, NF-kappaB signaling Received: September 06, 2016 Accepted: December 26, 2016 Published: January 31, 2017 ABSTRACT The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.

Introduction Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES). Methods Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers. Results TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors. Conclusions Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0080-7) contains supplementary material, which is available to authorized users.

Until recently, inflammatory chemokines were viewed mainly as indispensable “gate keepers” of immunity and inflammation. However, updated research indicates that members of this chemokine sub-family are important constituents of the tumor microenvironment, having multifaceted tumor-promoting roles in cancer. A very large number of studies indicate that many of the inflammatory chemokines are exploited by the tumor cells for their own benefit, and are actually skewed to the pro-malignancy phenotype. The different chemokines may be simultaneously expressed at the tumor site, having overlapping but also distinct tumor-promoting impacts. In general (except for the axis of CXCR3 and its ligands, that acts as a “double-edged sword”), the inflammatory chemokines induce immune imbalance at primary tumors and metastatic sites, doing so by promoting the presence and activation of tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC) and/or T regulatory cells (Treg). In parallel, immune suppression is ensued due to inhibition of Th1 cells and cytotoxic T lymphocytes (CTL). The chemokines also elevate metastasis-related processes, such as angiogenesis and osteoclastogenesis in the bone. Furthermore, they act directly on the tumor cells, promoting their proliferation, migration and invasion properties. Obviously, not all chemokines have the same pro-malignancy roles; however, chemokines that share the same receptor tend to have much in common in terms of their pro-cancerous activities. Accordingly, we will describe the roles of inflammatory chemokines in malignancy by using a receptor-based categorization: (1) CXCR1 and CXCR2 with their ELR+ CXC chemokine ligands, primarily CXCL8 (IL-8) but also CXCL1 (MGSA, GROα) and CXCL5 (ENA-78); (2) CXCR3 with its non-ELR CXC chemokine ligands: CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-Tac); (3) CCR2 and its ligands, mainly CCL2 (MCP-1); (4) CCR5 and its corresponding chemokines, with major emphasis on CCL5 (RANTES) and CCL3 (MIP-1α). Based on the findings obtained so far, we propose that inflammatory chemokines and their receptors are attractive therapeutic targets in malignancy, and discuss the expected difficulties in translating such approaches in the clinic.