Cell softness renders cytotoxic T lymphocytes and T leukemic cells resistant to perforin-mediated killing.

Mechanical force contributes to perforin pore formation at immune synapses, thus facilitating the cytotoxic T lymphocytes (CTL)-mediated killing of tumor cells in a unidirectional fashion. How such mechanical cues affect CTL evasion of perforin-mediated autolysis remains unclear. Here we show that activated CTLs use their softness to evade perforin-mediated autolysis, which, however, is shared by T leukemic cells to evade CTL killing. Downregulation of filamin A is identified to induce softness via ZAP70-mediated YAP Y357 phosphorylation and activation. Despite the requirements of YAP in both cell types for softness induction, CTLs are more resistant to YAP inhibitors than malignant T cells, potentially due to the higher expression of the drug-resistant transporter, MDR1, in CTLs. As a result, moderate inhibition of YAP stiffens malignant T cells but spares CTLs, thus allowing CTLs to cytolyze malignant cells without autolysis. Our findings thus hint a mechanical force-based immunotherapeutic strategy against T cell leukemia.

Tumor cell-released kynurenine biases MEP differentiation into megakaryocytes in individuals with cancer by activating AhR-RUNX1.

Tumor-derived factors are thought to regulate thrombocytosis and erythrocytopenia in individuals with cancer; however, such factors have not yet been identified. Here we show that tumor cell-released kynurenine (Kyn) biases megakaryocytic-erythroid progenitor cell (MEP) differentiation into megakaryocytes in individuals with cancer by activating the aryl hydrocarbon receptor-Runt-related transcription factor 1 (AhR-RUNX1) axis. During tumor growth, large amounts of Kyn from tumor cells are released into the periphery, where they are taken up by MEPs via the transporter SLC7A8. In the cytosol, Kyn binds to and activates AhR, leading to its translocation into the nucleus where AhR transactivates RUNX1, thus regulating MEP differentiation into megakaryocytes. In addition, activated AhR upregulates SLC7A8 in MEPs to induce positive feedback. Importantly, Kyn-AhR-RUNX1-regulated MEP differentiation was demonstrated in both humanized mice and individuals with cancer, providing potential strategies for the prevention of thrombocytosis and erythrocytopenia.

MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.

T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.

Cell Softness Prevents Cytolytic T-cell Killing of Tumor-Repopulating Cells.

Biomechanics is a fundamental feature of a cell. However, the manner by which actomysin tension affects tumor immune evasion remains unclear. Here we show that although cytotoxic T lymphocytes (CTL) can effectively destroy stiff differentiated tumor cells, they fail to kill soft tumor-repopulating cells (TRC). TRC softness prevented membrane pore formation caused by CTL-released perforin. Perforin interacting with nonmuscle myosin heavy-chain 9 transmitted forces to less F-actins in soft TRC, thus generating an inadequate contractile force for perforin pore formation. Stiffening TRC allowed perforin the ability to drill through the membrane, leading to CTL-mediated killing of TRC. Importantly, overcoming mechanical softness in human TRC also enhanced TRC cell death caused by human CTL, potentiating a mechanics-based immunotherapeutic strategy. These findings reveal a mechanics-mediated tumor immune evasion, thus potentially providing an alternative approach for tumor immunotherapy. SIGNIFICANCE: Tumor-repopulating cells evade CD8+ cytolytic T-cell killing through a mechanical softness mechanism, underlying the impediment of perforin pore formation at the immune synapse site.

Mechanisms by Which Dendritic Cells Present Tumor Microparticle Antigens to CD8+ T Cells.

Tumor cell-derived microparticles (T-MP) contain tumor antigen profiles as well as innate signals, endowing them with vaccine potential; however, the precise mechanism by which DCs present T-MP antigens to T cells remains unclear. Here, we show that T-MPs activate a lysosomal pathway that is required for DCs presenting tumor antigens of T-MPs. DCs endocytose T-MPs to lysosomes, where T-MPs increase lysosomal pH from 5.0 to a peak of 8.5 via NOX2-catalyzed reactive oxygen species (ROS) production. This increased pH, coupled with T-MP-driven lysosomal centripetal migration, promotes the formation of MHC class I-tumor antigen peptide complexes. Concurrently, endocytosis of T-MPs results in the upregulation of CD80 and CD86. T-MP-increased ROS activate lysosomal Ca2+ channel Mcoln2, leading to Ca2+ release. Released Ca2+ activates transcription factor EB (TFEB), a lysosomal master regulator that directly binds to CD80 and CD86 promoters, promoting gene expression. These findings elucidate a pathway through which DCs efficiently present tumor antigen from T-MPs to CD8+ T cells, potentiating T-MPs as a novel tumor cell-free vaccine with clinical applications. Cancer Immunol Res; 6(9); 1057-68. ©2018 AACR.

Publisher Correction: Chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward M1 phenotype.

In the originally published version of this Article, images in Fig. 5n were inadvertently replaced with duplicates of images in Fig. 5o during the production process. This has now been corrected in both the PDF and HTML versions of the Article.

Fibrin Stiffness Mediates Dormancy of Tumor-Repopulating Cells via a Cdc42-Driven Tet2 Epigenetic Program.

Dormancy is recognized as a critical biological event for tumorigenic cells surviving in an extremely harsh environment. Understanding the molecular process of dormancy can unlock novel approaches to tackle cancers. We recently reported that stem-like tumor-repopulating cells (TRC) sense mechanical signals and rapidly proliferate in a 90 Pa soft fibrin matrix. Here, we show that a stiff mechanical environment induces TRC dormancy via an epigenetic program initiated by translocation of Cdc42, a cytosolic regulator of mechanotransduction, into the nucleus, where it promotes transcription of hydroxymethylating enzyme Tet2. Tet2 epigenetically activated cell-cycle-inhibiting genes p21 and p27 to induce dormancy, but also caused downregulation of integrin ß3 to maintain dormancy. This stiffness-mediated dormancy was recapitulated in mouse models for both murine and primary human melanoma TRCs. These data identify an epigenetic program directed by mechanics, which drives highly tumorigenic TRCs to enter dormancy in a stiff mechanical environment.Significance: A mechanics-directed epigenetic program enables tumor-repopulating cells to enter dormancy in a stiff mechanical environment. Cancer Res; 78(14); 3926-37. ©2018 AACR.

Chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward M1 phenotype.

Resetting tumor-associated macrophages (TAMs) is a promising strategy to ameliorate the immunosuppressive tumor microenvironment and improve innate and adaptive antitumor immunity. Here we show that chloroquine (CQ), a proven anti-malarial drug, can function as an antitumor immune modulator that switches TAMs from M2 to tumor-killing M1 phenotype. Mechanistically, CQ increases macrophage lysosomal pH, causing Ca2+ release via the lysosomal Ca2+ channel mucolipin-1 (Mcoln1), which induces the activation of p38 and NF-κB, thus polarizing TAMs to M1 phenotype. In parallel, the released Ca2+ activates transcription factor EB (TFEB), which reprograms the metabolism of TAMs from oxidative phosphorylation to glycolysis. As a result, CQ-reset macrophages ameliorate tumor immune microenvironment by decreasing immunosuppressive infiltration of myeloid-derived suppressor cells and Treg cells, thus enhancing antitumor T-cell immunity. These data illuminate a previously unrecognized antitumor mechanism of CQ, suggesting a potential new macrophage-based tumor immunotherapeutic modality.

Tumor-Repopulating Cells Induce PD-1 Expression in CD8+ T Cells by Transferring Kynurenine and AhR Activation.

Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating T cells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8+ T cells through a transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) pathway. Interferon-γ produced by CD8+ T cells stimulates release of high levels of Kyn produced by TRCs, which is transferred into adjacent CD8+ T cells via the transporters SLC7A8 and PAT4. Kyn induces and activates AhR and thereby upregulates PD-1 expression. This Kyn-AhR pathway is confirmed in both tumor-bearing mice and cancer patients and its blockade enhances antitumor adoptive T cell therapy efficacy. Thus, we uncovered a mechanism of PD-1 upregulation with potential tumor immunotherapeutic applications.

STAT3/p53 pathway activation disrupts IFN-ß-induced dormancy in tumor-repopulating cells.

Dynamic interaction with the immune system profoundly regulates tumor cell dormancy. However, it is unclear how immunological cues trigger cancer cell-intrinsic signaling pathways for entering into dormancy. Here, we show that IFN-ß treatment induced tumor-repopulating cells (TRC) to enter dormancy through an indolamine 2,3-dioxygenase/kynurenine/aryl hydrocarbon receptor/p27-dependent (IDO/Kyn/AhR/p27-dependent) pathway. Strategies to block this metabolic circuitry did not relieve dormancy, but led to apoptosis of dormant TRCs in murine and human melanoma models. Specifically, blocking AhR redirected IFN-ß signaling to STAT3 phosphorylation through both tyrosine and serine sites, which subsequently facilitated STAT3 nuclear translocation and subsequent binding to the p53 promoter in the nucleus. Upregulation of p53 in turn disrupted the pentose phosphate pathway, leading to excessive ROS production and dormant TRC death. Additionally, in melanoma patients, high expression of IFN-ß correlated with tumor cell dormancy. Identification of this mechanism for controlling TRC dormancy by IFN-ß provides deeper insights into cancer-immune interaction and potential new cancer immunotherapeutic modalities.

Naturally occurring human phosphorylcholine antibodies are predominantly products of affinity-matured B cells in the adult.

Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM(+) memory subset, although significant numbers also were detected among naive, IgG(+), and CD27(+)CD43(+) B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.

Effect of biological therapy on levels of atheroprotective antibodies against phosphorylcholine and apolipoproteins in rheumatoid arthritis - a one year study.

OBJECTIVES: To examine how treatment of rheumatoid arthritis (RA) with anti-tumour necrosis factor alpha antagonists (anti-TNF) and B-cell targeting rituximab influences novel markers of atherosclerosis and inflammation, such as atheroprotective natural IgM antibodies against phosphorylcholine (anti-PC), oxidised low-density lipoprotein (oxLDL) and apolipoproteins. METHODS: In a prospective study 215 patients with RA were enrolled of whom 85.6% were seropositive, aged 57.9 ± 12.4 years, with mean disease duration 8.5 (5-15) years. 162 patients were treated with anti-TNF and 53 with rituximab for one year. The patients were assessed and blood sampled at 0, 3, 6 and 12 months. IgM anti-PC and oxLDL were determined by ELISA and apolipoproteins by immunoturbidimetry. RESULTS: IgM anti-PC increased by 26% during anti-TNF treatment, p<0.001, while decreased by 14% on rituximab, p=0.023, after 12 months of treatment. Patients in remission after 12 months, DAS28<2.6, had higher baseline anti-PC levels compared with those not in remission in both anti-TNF, p=0.007, and rituximab-treated subjects, p=0.041. In both treatment groups, levels of oxLDL increased temporarily at three months but apoA1 improved throughout the study. This effect was inversely correlated with changes in disease activity. The apoB and apoB/apoA1-ratio remained stable throughout the whole study period. CONCLUSIONS: Anti-TNF treatment demonstrated a favourable long-term effect on anti-PC levels. Low levels of IgM anti-PC may identify immune-deficient state and predict inferior therapy response. Biological therapies increased the level of the anti-atherogenic lipid apoA1. The impact of these effects on future CVD events deserves further studies.

1,5-anhydro-D-fructose and its derivatives: biosynthesis, preparation and potential medical applications.

1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with α-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.

Low levels of antibodies against phosphorylcholine predict development of stroke in a population-based study from northern Sweden.

BACKGROUND AND PURPOSE: Natural immunoglobulin M antibodies specific for phosphorylcholine (anti-PC) have been implicated in atherosclerosis. We have shown previously that high levels of anti-PC predict a slower progression of atherosclerosis in humans and that low levels of anti-PC are associated with higher risk for cardiovascular disease. Here we determine the association between anti-PC and the incidence of stroke. METHODS: Using a nested case control study design, we examined 227 incident cases (125 men and 102 women) of first-time stroke and 455 age- and sex-matched controls identified during a 13-year time period (1985 to 1999) within the population-based cohorts of the Västerbotten Intervention Project (VIP) and the World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease (WHO MONICA) project in Northern Sweden. Odds ratios of stroke with 95% CIs with adjustments for age, gender, smoking, serum cholesterol, diabetes, body mass index, and blood pressure were determined. Anti-PC levels were measured using ELISA. RESULTS: A significant association between low levels of anti-PC at baseline and incident stroke was seen for the whole group of anti-PC levels below the 30th percentile (multivariately adjusted odds ratio, 1.62; CI, 1.11 to 2.35). Analyses of gender-specific associations indicated fairly strong associations for females, especially at the lowest 30th percentile (multivariately adjusted odds ratio, 2.65; CI, 1.41 to 4.95). No associations were noted for men. CONCLUSION: Low anti-PC is a novel independent risk marker for development of stroke. Measurements of anti-PC could be used to identify immunodeficient subjects at an increased risk for stroke. The possibility that such subjects might be targets for novel modes of treatment such as immunotherapies deserves further investigation.