Metabolic Changes Associated With Cardiomyocyte Dedifferentiation Enable Adult Mammalian Cardiac Regeneration.

BACKGROUND: Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. METHODS: We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo. RESULTS: Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. CONCLUSIONS: Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

Comparison of the Diagnostic Accuracies of Monocyte Distribution Width, Procalcitonin, and C-Reactive Protein for Sepsis: A Systematic Review and Meta-Analysis.

OBJECTIVES: We performed a systemic review and meta-analysis to evaluate the diagnostic accuracy of monocyte distribution width (MDW) and to compare with procalcitonin and C-reactive protein (CRP), in adult patients with sepsis. DATA SOURCES: A systematic literature search was performed in PubMed, Embase, and the Cochrane Library to identify all relevant diagnostic accuracy studies published before October 1, 2022. STUDY SELECTION: Original articles reporting the diagnostic accuracy of MDW for sepsis detection with the Sepsis-2 or Sepsis-3 criteria were included. DATA EXTRACTION: Study data were abstracted by two independent reviewers using a standardized data extraction form. DATA SYNTHESIS: Eighteen studies were included in the meta-analysis. The pooled sensitivity and specificity of MDW were 84% (95% CI [79-88%]) and 68% (95% CI [60-75%]). The estimated diagnostic odds ratio and the area under the summary receiver operating characteristic curve (SROC) were 11.11 (95% CI [7.36-16.77]) and 0.85 (95% CI [0.81-0.89]). Significant heterogeneity was observed among the included studies. Eight studies compared the diagnostic accuracies of MDW and procalcitonin, and five studies compared the diagnostic accuracies of MDW and CRP. For MDW versus procalcitonin, the area under the SROC was similar (0.88, CI = 0.84-0.93 vs 0.82, CI = 0.76-0.88). For MDW versus CRP, the area under the SROC was similar (0.88, CI = 0.83-0.93 vs 0.86, CI = 0.78-0.95). CONCLUSIONS: The results of the meta-analysis indicate that MDW is a reliable diagnostic biomarker for sepsis as procalcitonin and CRP. Further studies investigating the combination of MDW and other biomarkers are advisable to increase the accuracy in sepsis detection.

Drug-Drug Interactions With Cyclosporine in the Anti-Hepatitis C Viral PrOD Combination Regimen of Paritaprevir/Ritonavir-Ombitasvir and Dasabuvir in Organ Transplant Recipients With Severe Hepatic Fibrosis or Cirrhosis.

BACKGROUND: The clinical guidelines suggest that the dosing of cyclosporine (CsA), during combination therapy with paritaprevir/ritonavir-ombitasvir and dasabuvir (PrOD), would be only one-fifth of the pre-PrOD total daily dose to be administered once daily. However, this dosing may not be applicable to all patients depending on their clinical condition. This study focuses on the pharmacokinetic dynamics of PrOD with CsA in Asian organ transplant recipients with severe liver fibrosis or cirrhosis who undergo concurrent treatment with PrOD treatment and CsA. The efficacy and safety of PrOD treatment was also evaluated. METHODS: Data from 7 patients obtained between January 2017 and September 2017 were retrospectively analyzed. Determinations of the blood concentrations of CsA were made, whether used as a single treatment or in combination therapy with PrOD. RESULTS: The combination regimen compared with CsA administered alone resulted in a 4.53-fold and 5.52-fold increase in the area under the concentration-time curve from time 0-12 hours (AUC0-12 h) of CsA on days 1 and 15, respectively. In addition, the maximal concentration, time to maximum concentration, and terminal phase elimination half-life (t1/2) of CsA were increased during the combined treatment of PrOD and CsA. The authors proposed reducing the CsA dosage during PrOD treatment to one-seventh of that of the pre-PrOD treatment of the total daily dose to maintain target CsA levels. All patients achieved sustained virologic responses at week 12. There were no episodes of serious adverse events or graft rejections observed. CONCLUSIONS: Although the combination with PrOD significantly affects the pharmacokinetics of CsA, it is effective and safe with regular monitoring of the CsA blood concentrations and appropriate CsA dose adjustment.

Cutting Edge: Intestinal Mucus Limits the Clonal Deletion of Developing T Cells Specific for an Oral Antigen.

A layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium. The MUC2 mucin is the primary constituent of intestinal mucus and plays critical protective roles against luminal microbes and other noxious agents. In this study, we investigated whether MUC2 helps maintain CD8 T cell tolerance toward intestinal luminal Ags by gavaging wild-type and Muc2-/- mice with a model Ag and monitoring immune responses posttreatment. We report that orally delivered OVA rapidly disseminates through the blood of Muc2-/- (but not control) mice and causes immune activation of Ag-specific CD8 T cells at both local and distal sites. Further, the administration of oral OVA to Muc2-/- mice led to its presentation by thymic dendritic cells and the deletion of Ag-specific thymocytes. Collectively, our findings suggest that intestinal mucus helps limit the shaping of the TCR repertoire of developing thymocytes by intestinal luminal Ags.

Duchenne muscular dystrophy newborn screening: the first 50,000 newborns screened in Taiwan.

BACKGROUND: Duchenne muscular dystrophy (DMD/Duchenne) is a progressive X-linked muscular disease with an overall incidence of 1:5,000 live male births. Recent availability in treatment for DMD raised the need of early diagnosis, and DMD became as a selective item of newborn screening (NBS) since Feb. 2021 in our center. MATERIALS AND METHODS: Dried blood spots (DBS) muscle-type creatine kinase (CK) isoform was measured with a commercialized kit with age-adjusted cutoffs. Subjects with an elevation of CK in the first screen were requested for a re-screen 2 weeks later. A DBS whole-exome sequencing (WES) panel for dystrophin and other neuromuscular-related genes was applied to confirm the diagnosis for subjects with persistent hyperCKemia. RESULTS: During a 1-year period, 50,572 newborns (male 26,130) received DMD screening at a mean age of 2 days (SD 1 day). Among them, 632 (1.2%) had an elevated CK value. A re-screen at a mean age of 14 days (SD 8 days) revealed 14 subjects with persistent hyperCKemia, and DMD was confirmed in 3 of them. The incidence of DMD in Taiwan was 1:8,710 (95% CI 1 in 2,963 to 1 in 25,610) live birth males. Results of DMD DBS also assisted in Pompe newborn screening. CONCLUSIONS: NBS for DMD enables earlier management of the disease. The high re-screening rate could potentially be waived by moving the DBS WES assay to a second-tier test. The long-term benefit and the impact of newborn screening on the prognosis of DMD, however, remain further elucidated.

Incisor liability and its effects among East Asian children.

BACKGROUND/PURPOSE: Incisor liability is the discrepancy in the sum of the mesiodistal crown width between the primary and permanent incisors. Incisor liability affects the integrity and eruption of the permanent incisors during the transition from the primary to permanent dentition. This study investigated the incisor liability in the primary dentition of Taiwanese children. METHODS: The digital periapical films of 203 upper arches of 105 boys and 98 girls and 195 lower arches of 119 boys and 76 girls aged between 3 and 6 years were selected in this retrospective study. The mesiodistal crown widths of the primary and permanent incisors were measured using the medical imaging software for both arches. Differences in incisor liability values were statistically analyzed. RESULTS: The mean ± standard deviation of the incisor liability values were 8.32 ± 1.88 and 6.91 ± 1.13 mm for the upper and lower arches, respectively, in all children. The incisor liability was closely related with the total crown widths of the permanent incisors for upper and lower arches. The incisor liability values were higher among boys than girls for the upper but not lower arch. CONCLUSION: Incisor liability differs depending on ethnicity. In Taiwanese children, incisor liability was closely related with the crown widths of the permanent incisors. The incisor liability values of boys were higher than those of girls in the upper arch but not the lower arch.

Folinate Supplementation Ameliorates Methotrexate Induced Mitochondrial Formate Depletion In Vitro and In Vivo.

(1) Background: Antifolate methotrexate (MTX) is the most common disease-modifying antirheumatic drug (DMARD) for treating human rheumatoid arthritis (RA). The mitochondrial-produced formate is essential for folate-mediated one carbon (1C) metabolism. The impacts of MTX on formate homeostasis in unknown, and rigorously controlled kinetic studies can greatly help in this regard. (2) Methods: Combining animal model (8-week old female C57BL/6JNarl mice, n = 18), cell models, stable isotopic tracer studies with gas chromatography/mass spectrometry (GC/MS) platforms, we systematically investigated how MTX interferes with the partitioning of mitochondrial and cytosolic formate metabolism. (3) Results: MTX significantly reduced de novo deoxythymidylate (dTMP) and methionine biosyntheses from mitochondrial-derived formate in cells, mouse liver, and bone marrow, supporting our postulation that MTX depletes mitochondrial 1C supply. Furthermore, MTX inhibited formate generation from mitochondria glycine cleavage system (GCS) both in vitro and in vivo. Folinate selectively rescued 1C metabolic pathways in a tissue-, cellular compartment-, and pathway-specific manner: folinate effectively reversed the inhibition of mitochondrial formate-dependent 1C metabolism in mouse bone marrow (dTMP, methionine, and GCS) and cells (dTMP and GCS) but not methionine synthesis in liver/liver-derived cells. Folinate failed to fully recover hepatic mitochondrial-formate utilization for methionine synthesis, suggesting that the efficacy of clinical folinate rescue in MTX therapy on hepatic methionine metabolism is poor. (4) Conclusion: Conducting studies in mouse and cell models, we demonstrate novel findings that MTX specifically depletes mitochondrial 1C supply that can be ameliorated by folinate supplementation except for hepatic transmethylation. These results imply that clinical use of low-dose MTX may particularly impede 1C metabolism via depletion of mitochondrial formate. The MTX induced systematic and tissue-specific formate depletion needs to be addressed more carefully, and the efficacy of folinate with respect to protecting against such depletion deserves to be evaluated in medical practice.

A global phylogeny of Stegnogramma ferns (Thelypteridaceae): generic and sectional revision, historical biogeography and evolution of leaf architecture.

The thelypteroid fern genus Stegnogramma s.l. contains around 18-35 species and has a global, cross-continental distribution ranging from tropical to temperate regions. Several genera and infrageneric sections have been recognized previously in Stegnogramma s.l., but their phylogenetic relationships are still unclear. In this study, we present a global phylogeny of Stegnogramma s.l. with the most comprehensive sampling to date and aim to pinpoint the phylogenetic positions of biogeographically and taxonomically important taxa. Based on the reconstructed historical biogeography and character evolution, we propose a new (infra)generic classification and discuss the diversification of Stegnogramma s.l. in a biogeographical context. New names or combinations are made for 12 (infra)species, including transferring the monotypic species of Craspedosorus to Leptogramma. Finally, we discuss a possible link between leaf architecture and ecological adaptation, and hypothesize that the increase in leaf dissection and free-vein proportion is an adaptive feature to cool climates in Stegnogramma s.l.

Cancer-associated fibroblast-derived interleukin-1ß activates protumor C-C motif chemokine ligand 22 signaling in head and neck cancer.

Oral cancer, a subtype of head and neck cancer, is characterized by increased infiltrating regulatory T cells (Treg); however, the pathological significance of the increase in Tregs in disease prognosis and progression and their underlying mechanism remain unestablished. C-C motif chemokine ligand 22 (CCL22) has been implicated in the recruitment of Tregs. We used RT-qPCR to determine CCL22 mRNA expression in clinical specimens and cultured cells. Loss-of-function and gain-of-function studies were carried out to analyze the effects of CCL22 modulations on cell proliferation, migration, invasion, and tumorigenesis and the mechanism involved in the deregulation of CCL22. In oral cancer specimens, CCL22 mRNA was upregulated. The increase was not only associated with reduced disease-free survival but also strongly correlated with an increase in FOXP3 mRNA, a master regulator of Treg development and functions. Silencing CCL22 expression reduced cell proliferation, migration, and invasion, whereas ectopic overexpression showed opposite effects. Manipulation of CCL22 expression in cancer cells altered tumorigenesis in both immune-compromised and -competent mice, supporting both autonomous and non-autonomous actions of CCL22. Release of interleukin 1ß (IL-1ß) from cancer-associated fibroblasts (CAF) induces CCL22 mRNA expression in oral cancer cells by activating transcription factor nuclear factor kappa B (NF-κB). Our data support a model in which CAF-derived IL-1ß, CCL22, and its receptor CCR4 foster a protumor environment by promoting cell transformation and Treg infiltration. Intervention of the IL-1ß-CCL22-CCR4 signaling axis may offer a novel therapeutic strategy for oral cancer treatment.

Modeling the CH Stretch/Torsion/Rotation Couplings in Methyl Peroxy (CH3OO).

The manifestations of CH stretch/torsion/rotation coupling in the region of the CH stretch fundamentals are explored in the CH3OO radical. Following our earlier study of the fundamental in the totally symmetric CH stretch (the ν2 fundamental), this work focuses on the other two CH stretch fundamentals, ν1 and ν9, which would be degenerate in the absence of a barrier in the potential along the methyl torsion coordinate. The simplest model, which assumes a decoupling of the CH stretch vibrations from the torsion, fails to reproduce several important features of the spectrum. Specifically, the absence of a strong peak around the origin of the ν1 fundamental and broadening of the strong peak near the origin in the observed spectrum of the ν9 fundamental are not captured by this model. The origins of these features are explored through two more sophisticated treatments of the torsion/CH stretch couplings. In the first, a four-dimensional potential based on the three CH stretches and the torsion is developed and shown to reproduce both of these features. On the basis of the results of these calculations, the calculated parameters are adjusted to simulate the recorded spectrum. To further explore the torsion/CH stretch couplings in CH3OO, a 9-state model Hamiltonian is developed and discussed. The implications of various types of couplings on the observed energy level patterns are also discussed.

SLAM-SAP signaling promotes differentiation of IL-17-producing T cells and progression of experimental autoimmune encephalomyelitis.

IL-17 plays critical roles in host defenses, combating bacterial and fungal infections, as well as the pathogenesis of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). The signaling adaptor SAP is essential for normal immune homeostasis and mutations within SH2D1A, the locus encoding this protein, result in serious and sometimes fatal syndromes, including X-linked lymphoproliferative disease and severe cases of common variable immunodeficiency. However, the precise cellular basis of how SAP deficiency contributes to immune dysfunction remains incompletely understood. In this study, we found that CD4 and CD8 T cells lacking SAP had a diminished capacity to differentiate into IL-17-producing Th17 and T cytotoxic (Tc17) cells relative to wild-type lymphocytes. The use of costimulating SLAM Abs was found to augment the differentiation of IL-17-secreting effectors in wild-type but not Sh2d1a(-/-) splenic T cells under IL-17-polarizing conditions. In addition, SAP's regulation of IL-17-secreting T cells was shown to be a T cell-intrinsic role, as purified naive Sh2d1a(-/-) CD4 and CD8 T cells were inherently defective at converting into Th17 and Tc17 cells in vitro and in vivo. Furthermore, Sh2d1a(-/-) mice were protected from EAE and exhibited greatly decreased numbers of CNS-infiltrating Th17 and Tc17 effector T cells and reduced disease severity. Collectively, these results suggest that SLAM-SAP signaling drives the differentiation and function of Th17 and Tc17 cells in vitro and in vivo and contributes to the pathogenesis of autoimmunity in EAE.

Manifestations of Torsion-CH Stretch Coupling in the Infrared Spectrum of CH3OO.

With a step-scan Fourier-transform spectrometer we recorded temporally resolved infrared absorption spectra of CH3OO radicals that were produced upon irradiation of CH3COCH3 and O2 at 193 nm in a flowing mixture. At a resolution of 0.15 cm(-1), the rotational structure of the ν2 band of CH3OO near 2954.4 cm(-1) is partially resolved and shows an unexpectedly broadened, and somewhat distorted, Q-branch. A 4D model Hamiltonian, consisting of three CH stretches and the methyl torsion, was developed to explore the origins of this broadening. The vibrational progressions predicted by the model Hamiltonian and the rotational contours of the ν2 band, based on experimental ground-state rotational parameters and their values scaled by their calculated ratios for the upper state, produced simulations in satisfactory agreement with the observed spectrum. These results provide new insight into the vibrational couplings in CH3OO.

Infrared absorption spectrum of the simplest deuterated Criegee intermediate CD2OO.

We report a transient infrared (IR) absorption spectrum of the simplest deuterated Criegee intermediate CD2OO recorded using a step-scan Fourier-transform spectrometer coupled with a multipass absorption cell. CD2OO was produced from photolysis of flowing mixtures of CD2I2, N2, and O2 (13 or 87 Torr) with laser light at 308 nm. The recorded spectrum shows close structural similarity with the spectrum of CH2OO reported previously [Y.-T. Su et al., Science 340, 174 (2013)]. The four bands observed at 852, 1017, 1054, and 1318 cm(-1) are assigned to the OO stretching mode, two distinct in-plane OCD bending modes, and the CO stretching mode of CD2OO, respectively, according to vibrational wavenumbers, IR intensities, rotational contours, and deuterium-isotopic shifts predicted with extensive quantum-chemical calculations. The CO-stretching mode of CD2OO at 1318 cm(-1) is blue shifted from the corresponding band of CH2OO at 1286 cm(-1); this can be explained by a mechanism based on mode mixing and isotope substitution. A band near 936 cm(-1), observed only at higher pressure (87 Torr), is tentatively assigned to the CD2 wagging mode of CD2IOO.

Infrared spectrum of the simplest Criegee intermediate CH2OO at resolution 0.25 cm(-1) and new assignments of bands 2ν9 and ν5.

The simplest Criegee intermediate CH2OO is important in atmospheric chemistry. It has been detected in the reaction of CH2I + O2 with various spectral methods, including infrared spectroscopy; infrared absorption of CH2OO was recorded at resolution 1.0 cm(-1) in our laboratory. We have improved our system and recorded the infrared spectrum of CH2OO at resolution 0.25 cm(-1) with rotational structures partially resolved. Observed vibrational wavenumbers and relative intensities are improved from those of the previous report and agree well with those predicted with quantum-mechanical calculations using the MULTIMODE method on an accurate potential energy surface. Observed rotational structures also agree with the simulated spectra according to theoretical predictions. In addition to derivation of critical vibrational and rotational parameters of the vibrationally excited states to confirm the assignments, the spectrum with improved resolution provides new assignments for bands 2ν9 at 1234.2 cm(-1) and ν5 at 1213.3 cm(-1); some hot bands and combination bands are also tentatively assigned.

Concentration effects of grape seed extracts in anti-oral cancer cells involving differential apoptosis, oxidative stress, and DNA damage.

BACKGROUND: Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer. METHODS: The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage. RESULTS: High concentrations (50-400 µg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 µg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations. CONCLUSIONS: Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.

Infrared absorption of gaseous CH2BrOO detected with a step-scan Fourier-transform absorption spectrometer.

CH2BrOO radicals were produced upon irradiation, with an excimer laser at 248 nm, of a flowing mixture of CH2Br2 and O2. A step-scan Fourier-transform spectrometer coupled with a multipass absorption cell was employed to record temporally resolved infrared (IR) absorption spectra of reaction intermediates. Transient absorption with origins at 1276.1, 1088.3, 961.0, and 884.9 cm(-1) are assigned to ν4 (CH2-wagging), ν6 (O-O stretching), ν7 (CH2-rocking mixed with C-O stretching), and ν8 (C-O stretching mixed with CH2-rocking) modes of syn-CH2BrOO, respectively. The assignments were made according to the expected photochemistry and a comparison of observed vibrational wavenumbers, relative IR intensities, and rotational contours with those predicted with the B3LYP/aug-cc-pVTZ method. The rotational contours of ν7 and ν8 indicate that hot bands involving the torsional (ν12) mode are also present, with transitions 7(0)(1)12(v)(v) and 8(0)(1)12(v)(v), v = 1-10. The most intense band (ν4) of anti-CH2BrOO near 1277 cm(-1) might have a small contribution to the observed spectra. Our work provides information for directly probing gaseous CH2BrOO with IR spectroscopy, in either the atmosphere or laboratory experiments.

Clinical Characteristics, Genetic Features, and Long-Term Outcome of Wilson's Disease in a Taiwanese Population: An 11-Year Follow-Up Study.

OBJECTIVE: aaWilson's disease (WD) is a rare genetic disorder of copper metabolism, and longitudinal follow-up studies are limited. We performed a retrospective analysis to determine the clinical characteristics and long-term outcomes in a large WD cohort. METHODS: aaMedical records of WD patients diagnosed from 2006-2021 at National Taiwan University Hospital were retrospectively evaluated for clinical presentations, neuroimages, genetic information, and follow-up outcomes. RESULTS: aaThe present study enrolled 123 WD patients (mean follow-up: 11.12 ± 7.41 years), including 74 patients (60.2%) with hepatic features and 49 patients (39.8%) with predominantly neuropsychiatric symptoms. Compared to the hepatic group, the neuropsychiatric group exhibited more Kayser-Fleischer rings (77.6% vs. 41.9%, p < 0.01), lower serum ceruloplasmin levels (4.9 ± 3.9 vs. 6.3 ± 3.9 mg/dL, p < 0.01), smaller total brain and subcortical gray matter volumes (p < 0.0001), and worse functional outcomes during follow-up (p = 0.0003). Among patients with available DNA samples (n = 59), the most common mutations were p.R778L (allelic frequency of 22.03%) followed by p.P992L (11.86%) and p.T935M (9.32%). Patients with at least one allele of p.R778L had a younger onset age (p = 0.04), lower ceruloplasmin levels (p < 0.01), lower serum copper levels (p = 0.03), higher percentage of the hepatic form (p = 0.03), and a better functional outcome during follow-up (p = 0.0012) compared to patients with other genetic variations. CONCLUSION: aaThe distinct clinical characteristics and long-term outcomes of patients in our cohort support the ethnic differences regarding the mutational spectrum and clinical presentations in WD.

Tumor-derived GCSF Alters Tumor and Systemic Immune System Cell Subset Composition and Signaling.

While immunotherapies such as immune checkpoint blockade and adoptive T-cell therapy improve survival for a subset of human malignancies, many patients fail to respond. Phagocytes including dendritic cells (DC), monocytes, and macrophages (MF) orchestrate innate and adaptive immune responses against tumors. However, tumor-derived factors may limit immunotherapy effectiveness by altering phagocyte signal transduction, development, and activity. Using Cytometry by Time-of-Flight, we found that tumor-derived GCSF altered myeloid cell distribution both locally and systemically. We distinguished a large number of GCSF-induced immune cell subset and signal transduction pathway perturbations in tumor-bearing mice, including a prominent increase in immature neutrophil/myeloid-derived suppressor cell (Neut/MDSC) subsets and tumor-resident PD-L1+ Neut/MDSCs. GCSF expression was also linked to distinct tumor-associated MF populations, decreased conventional DCs, and splenomegaly characterized by increased splenic progenitors with diminished DC differentiation potential. GCSF-dependent dysregulation of DC development was recapitulated in bone marrow cultures in vitro, using medium derived from GCSF-expressing tumor cell cultures. Importantly, tumor-derived GCSF impaired T-cell adoptive cell therapy effectiveness and was associated with increased tumor volume and diminished survival of mice with mammary cancer. Treatment with neutralizing anti-GCSF antibodies reduced colonic and circulatory Neut/MDSCs, normalized colonic immune cell composition and diminished tumor burden in a spontaneous model of mouse colon cancer. Analysis of human colorectal cancer patient gene expression data revealed a significant correlation between survival and low GCSF and Neut/MDSC gene expression. Our data suggest that normalizing GCSF bioactivity may improve immunotherapy in cancers associated with GCSF overexpression. Significance: Tumor-derived GCSF leads to systemic immune population changes. GCSF blockade restores immune populations, improves immunotherapy, and reduces tumor size, paralleling human colorectal cancer data. GCSF inhibition may synergize with current immunotherapies to treat GCSF-secreting tumors.

RasGRP1 regulates antigen-induced developmental programming by naive CD8 T cells.

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1(-/-) 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.