In patients with cancer, the wasting syndrome, cachexia, is associated with caloric deficiency. Here, we describe tumor-induced alterations of the host metabolic response to caloric deficiency that ...cause intratumoral immune suppression. In pre-cachectic mice with transplanted colorectal cancer or autochthonous pancreatic ductal adenocarcinoma (PDA), we find that IL-6 reduces the hepatic ketogenic potential through suppression of PPARalpha, the transcriptional master regulator of ketogenesis. When these mice are challenged with caloric deficiency, the resulting relative hypoketonemia triggers a marked rise in glucocorticoid levels. Multiple intratumoral immune pathways are suppressed by this hormonal stress response. Moreover, administering corticosterone to elevate plasma corticosterone to a level that is lower than that occurring in cachectic mice abolishes the response of mouse PDA to an immunotherapy that has advanced to clinical trials. Therefore, tumor-induced IL-6 impairs the ketogenic response to reduced caloric intake, resulting in a systemic metabolic stress response that blocks anti-cancer immunotherapy.
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•IL-6 suppresses hepatic ketogenesis in pre-cachectic, tumor-bearing mice•During caloric deficiency, hypoketonemia triggers marked glucocorticoid secretion•Glucocorticoids, induced by metabolic stress, suppress intratumoral immunity•Stress-induced glucocorticoids cause failure of cancer immunotherapy
Flint and Janowitz et al. reveal the intricate links between cancer cachexia, hepatic metabolism, and tumor immunology. They find that tumor-induced IL-6 suppresses hepatic ketogenesis, and during caloric deficiency, this triggers marked glucocorticoid secretion. This hormonal stress response suppresses intratumoral immunity and causes failure of anti-cancer immunotherapy.
The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with ...immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.
An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer ...cell-specific CD8 ⁺ T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP ⁺ stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP ⁺ cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP ⁺ CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP ⁺ CAF, may direct tumor immune evasion in a model of human PDA.
Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that ...applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRɣ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%.
Fibroblastic reticular cells (FRCs), through their expression of CC chemokine ligand (CCL)19 and CCL21, attract and retain T cells in lymph nodes (LNs), but whether this function applies to both ...resting and activated T cells has not been examined. Here we describe a model for conditionally depleting FRCs from LNs based on their expression of the diphtheria toxin receptor (DTR) directed by the gene encoding fibroblast activation protein-α (FAP). As expected, depleting FAP ⁺ FRCs causes the loss of naïve T cells, B cells, and dendritic cells from LNs, and this loss decreases the magnitude of the B- and T-cell responses to a subsequent infection with influenza A virus. In contrast, depleting FAP ⁺ FRCs during an ongoing influenza infection does not diminish the number or continued response of activated T and B cells in the draining LNs, despite still resulting in the loss of naïve T cells. Therefore, different rules govern the LN trafficking of resting and activated T cells; once a T cell is engaged in antigen-specific clonal expansion, its retention no longer depends on FRCs or their chemokines, CCL19 and CCL21. Our findings suggest that activated T cells remain in the LN because they down-regulate the expression of the sphingosine-1 phosphate receptor-1, which mediates the exit of lymphocytes from secondary lymphoid organs. Therefore, LN retention of naïve lymphocytes and the initiation of an immune response depend on FRCs, but is an FRC independent and possibly cell-autonomous response of activated T cells, which allows the magnitude of clonal expansion to determine LN egress.
Effective immune responses depend on efficient antigen uptake in the periphery, transport of those antigens to, and presentation in draining lymph nodes (LNs). These processes have been studied ...intensively using stable fluorescent proteins (FPs) as model antigens. To date, ZsGreen is the only FP that can be tracked efficiently towards LNs, hence, it is difficult to compare studies using alternated tracking proteins. Here, we systematically compared six different FPs. We included ZsGreen, ZsYellow, DsRed, AsRed, mCherry, and mRFP based on sequence homology and/or origin species, and generated FP-expressing tumor cell lines. Stability of fluorescent signal was assessed in vitro over time, across different pH environments, and in vivo through FP antigen uptake and transfer to immune cells isolated from tumors and tumor-draining LNs. ZsGreen could be detected in high percentages of all analyzed tumor-infiltrating immune cells, with highest amounts in tumor-associated macrophages (TAMs) and type 2 conventional dendritic cells (cDC2s). ZsYellow, AsRed, and DsRed followed a similar pattern, but percentages of FP-containing immune cells in the tumor were lower than for ZsGreen. Strikingly, mRFP and mCherry demonstrated a 'non-canonical' antigen uptake pattern where percentages of FP-positive tumor-infiltrating immune cells were highest for cDC1s not TAMs and cDC2s despite comparable stabilities and localization of all FPs. Analysis of antigen-containing cells in the LN was hindered by intracellular degradation of FPs. Only ZsGreen could be efficiently tracked to the LN, though some signal was measurable for ZsYellow and DsRed. In summary, we find that detection of antigen uptake and distribution is subject to variabilities related to fluorophore nature. Future experiments need to consider that these processes might be impacted by protein expression, stability, or other unknown factors. Thus, our data sheds light on potential under-appreciated mechanisms regulating antigen transfer and highlights potential uses and necessary caveats to interpretation based on FP use.
Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a ...fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.
The RNA cap methyltransferase CMTR1 methylates the first transcribed nucleotide of RNA polymerase II transcripts, impacting gene expression mechanisms, including during innate immune responses. Using ...mass spectrometry, we identify a multiply phosphorylated region of CMTR1 (phospho-patch P-Patch), which is a substrate for the kinase CK2 (casein kinase II). CMTR1 phosphorylation alters intramolecular interactions, increases recruitment to RNA polymerase II, and promotes RNA cap methylation. P-Patch phosphorylation occurs during the G1 phase of the cell cycle, recruiting CMTR1 to RNA polymerase II during a period of rapid transcription and RNA cap formation. CMTR1 phosphorylation is required for the expression of specific RNAs, including ribosomal protein gene transcripts, and promotes cell proliferation. CMTR1 phosphorylation is also required for interferon-stimulated gene expression. The cap-snatching virus, influenza A, utilizes host CMTR1 phosphorylation to produce the caps required for virus production and infection. We present an RNA cap methylation control mechanism whereby CK2 controls CMTR1, enhancing co-transcriptional capping.
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•CK2 phosphorylates the RNA cap methyltransferase CMTR1, resulting in binding to RNA Pol II•CMTR1 P-Patch phosphorylation is required for RNA cap methylation and gene expression•Expression of interferon-stimulated genes (ISGs) is dependent on CMTR1 P-Patch phosphorylation•Influenza A, a cap-snatching virus, is dependent on CMTR1 phosphorylation for infection
Lukozek et al. report that the RNA cap methyltransferase CMTR1 is phosphorylated by CK2, which promotes the interaction with RNA Pol II and RNA cap formation. CMTR1 phosphorylation is required for ribosomal protein gene expression and the interferon response. Propagation of the cap-snatching influenza virus depends on host CMTR1 phosphorylation.
The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In ...this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.
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