Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

Six-dimensional dynamic tractography atlas of language connectivity in the developing brain.

During a verbal conversation, our brain moves through a series of complex linguistic processing stages: sound decoding, semantic comprehension, retrieval of semantically coherent words, and overt production of speech outputs. Each process is thought to be supported by a network consisting of local and long-range connections bridging between major cortical areas. Both temporal and extratemporal lobe regions have functional compartments responsible for distinct language domains, including the perception and production of phonological and semantic components. This study provides quantitative evidence of how directly connected inter-lobar neocortical networks support distinct stages of linguistic processing across brain development. Novel six-dimensional tractography was used to intuitively visualize the strength and temporal dynamics of direct inter-lobar effective connectivity between cortical areas activated during each linguistic processing stage. We analysed 3401 non-epileptic intracranial electrode sites from 37 children with focal epilepsy (aged 5-20 years) who underwent extra-operative electrocorticography recording. Principal component analysis of auditory naming-related high-gamma modulations determined the relative involvement of each cortical area during each linguistic processing stage. To quantify direct effective connectivity, we delivered single-pulse electrical stimulation to 488 temporal and 1581 extratemporal lobe sites and measured the early cortico-cortical spectral responses at distant electrodes. Mixed model analyses determined the effects of naming-related high-gamma co-augmentation between connecting regions, age, and cerebral hemisphere on the strength of effective connectivity independent of epilepsy-related factors. Direct effective connectivity was strongest between extratemporal and temporal lobe site pairs, which were simultaneously activated between sentence offset and verbal response onset (i.e. response preparation period); this connectivity was approximately twice more robust than that with temporal lobe sites activated during stimulus listening or overt response. Conversely, extratemporal lobe sites activated during overt response were equally connected with temporal lobe language sites. Older age was associated with increased strength of inter-lobar effective connectivity especially between those activated during response preparation. The arcuate fasciculus supported approximately two-thirds of the direct effective connectivity pathways from temporal to extratemporal auditory language-related areas but only up to half of those in the opposite direction. The uncinate fasciculus consisted of <2% of those in the temporal-to-extratemporal direction and up to 6% of those in the opposite direction. We, for the first time, provided an atlas which quantifies and animates the strength, dynamics, and direction specificity of inter-lobar neural communications between language areas via the white matter pathways. Language-related effective connectivity may be strengthened in an age-dependent manner even after the age of 5.

Clinical Features and Multiplatform Molecular Analysis Assist in Understanding Patient Response to Anti-PD-1/PD-L1 in Renal Cell Carcinoma.

Predicting response to ICI therapy among patients with renal cell carcinoma (RCC) has been uniquely challenging. We analyzed patient characteristics and clinical correlates from a retrospective single-site cohort of advanced RCC patients receiving anti-PD-1/PD-L1 monotherapy (N = 97), as well as molecular parameters in a subset of patients, including multiplexed immunofluorescence (mIF), whole exome sequencing (WES), T cell receptor (TCR) sequencing, and RNA sequencing (RNA-seq). Clinical factors such as the development of immune-related adverse events (odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.05-5.91) and immunological prognostic parameters, including a higher percentage of circulating lymphocytes (23.4% vs. 17.4%, p = 0.0015) and a lower percentage of circulating neutrophils (61.8% vs. 68.5%, p = 0.0045), correlated with response. Previously identified gene expression signatures representing pathways of angiogenesis, myeloid inflammation, T effector presence, and clear cell signatures also correlated with response. High PD-L1 expression (>10% cells) as well as low TCR diversity (≤644 clonotypes) were associated with improved progression-free survival (PFS). We corroborate previously published findings and provide preliminary evidence of T cell clonality impacting the outcome of RCC patients. To further biomarker development in RCC, future studies will benefit from integrated analysis of multiple molecular platforms and prospective validation.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Dynamic tractography: Integrating cortico-cortical evoked potentials and diffusion imaging.

INTRODUCTION: Cortico-cortical evoked potentials (CCEPs) are utilized to identify effective networks in the human brain. Following single-pulse electrical stimulation of cortical electrodes, evoked responses are recorded from distant cortical areas. A negative deflection (N1) which occurs 10-50 â€‹ms post-stimulus is considered to be a marker for direct cortico-cortical connectivity. However, with CCEPs alone it is not possible to observe the white matter pathways that conduct the signal or accurately predict N1 amplitude and latency at downstream recoding sites. Here, we develop a new approach, termed "dynamic tractography," which integrates CCEP data with diffusion-weighted imaging (DWI) data collected from the same patients. This innovative method allows greater insights into cortico-cortical networks than provided by each method alone and may improve the understanding of large-scale networks that support cognitive functions. The dynamic tractography model produces several fundamental hypotheses which we investigate: 1) DWI-based pathlength predicts N1 latency; 2) DWI-based pathlength negatively predicts N1 voltage; and 3) fractional anisotropy (FA) along the white matter path predicts N1 propagation velocity. METHODS: Twenty-three neurosurgical patients with drug-resistant epilepsy underwent both extraoperative CCEP recordings and preoperative DWI scans. Subdural grids of 3 â€‹mm diameter electrodes were used for stimulation and recording, with 98-128 eligible electrodes per patient. CCEPs were elicited by trains of 1 â€‹Hz stimuli with an intensity of 5 â€‹mA and recorded at a sample rate of 1 â€‹kHz. N1 peak and latency were defined as the maximum of a negative deflection within 10-50 â€‹ms post-stimulus with a z-score > 5 relative to baseline. Electrodes and DWI were coregistered to construct electrode connectomes for white matter quantification. RESULTS: Clinical variables (age, sex, number of anti-epileptic drugs, handedness, and stimulated hemisphere) did not correlate with the key outcome measures (N1 peak amplitude, latency, velocity, or DWI pathlength). All subjects and electrodes were therefore pooled into a group-level analysis to determine overall patterns. As hypothesized, DWI path length positively predicted N1 latency (R2 â€‹= â€‹0.81, ߠ​= â€‹1.51, p â€‹= â€‹4.76e-16) and negatively predicted N1 voltage (R2 â€‹= â€‹0.79, ߠ​= â€‹-0.094, p â€‹= â€‹9.30e-15), while FA predicted N1 propagation velocity (R2 â€‹= â€‹0.35, ߠ​= â€‹48.0, p â€‹= â€‹0.001). CONCLUSION: We have demonstrated that the strength and timing of the CCEP N1 is dependent on the properties of the underlying white matter network. Integrated CCEP and DWI visualization allows robust localization of intact axonal pathways which effectively interconnect eloquent cortex.

Four-dimensional map of direct effective connectivity from posterior visual areas.

Lower- and higher-order visual cortices in the posterior brain, ranging from the medial- and lateral-occipital to fusiform regions, are suggested to support visual object recognition, whereas the frontal eye field (FEF) plays a role in saccadic eye movements which optimize visual processing. Previous studies using electrophysiology and functional MRI techniques have reported that tasks requiring visual object recognition elicited cortical activation sequentially in the aforementioned posterior visual regions and FEFs. The present study aims to provide unique evidence of direct effective connectivity outgoing from the posterior visual regions by measuring the early component (10-50 â€‹ms) of cortico-cortical spectral responses (CCSRs) elicited by weak single-pulse direct cortical electrical stimulation. We studied 22 patients who underwent extraoperative intracranial EEG recording for clinical localization of seizure foci and functionally-important brain regions. We used animations to visualize the spatiotemporal dynamics of gamma band CCSRs elicited by stimulation of three different posterior visual regions. We quantified the strength of CCSR-defined effective connectivity between the lower- and higher-order posterior visual regions as well as from the posterior visual regions to the FEFs. We found that effective connectivity within the posterior visual regions was larger in the feedforward (i.e., lower-to higher-order) direction compared to the opposite direction. Specifically, connectivity from the medial-occipital region was largest to the lateral-occipital region, whereas that from the lateral-occipital region was largest to the fusiform region. Among the posterior visual regions, connectivity to the FEF was largest from the lateral-occipital region and the mean peak latency of CCSR propagation from the lateral-occipital region to FEF was 26 â€‹ms. Our invasive study of the human brain using a stimulation-based intervention supports the model that the posterior visual regions have direct cortico-cortical connectivity pathways in which neural activity is transferred preferentially from the lower-to higher-order areas. The human brain has direct cortico-cortical connectivity allowing a rapid transfer of neural activity from the lateral-occipital region to the FEF.

Quantitative analysis of intracranial electrocorticography signals using the concept of statistical parametric mapping.

Statistical parametric mapping (SPM) is a technique with which one can delineate brain activity statistically deviated from the normative mean, and has been commonly employed in noninvasive neuroimaging and EEG studies. Using the concept of SPM, we developed a novel technique for quantification of the statistical deviation of an intracranial electrocorticography (ECoG) measure from the nonepileptic mean. We validated this technique using data previously collected from 123 patients with drug-resistant epilepsy who underwent resective epilepsy surgery. We determined how the measurement of statistical deviation of modulation index (MI) from the non-epileptic mean (rated by z-score) improved the performance of seizure outcome classification model solely based on conventional clinical, seizure onset zone (SOZ), and neuroimaging variables. Here, MI is a summary measure quantifying the strength of in-situ coupling between high-frequency activity at >150 Hz and slow wave at 3-4 Hz. We initially generated a normative MI atlas showing the mean and standard deviation of slow-wave sleep MI of neighboring non-epileptic channels of 47 patients, whose ECoG sampling involved all four lobes. We then calculated 'MI z-score' at each electrode site. SOZ had a greater 'MI z-score' compared to non-SOZ in the remaining 76 patients. Subsequent multivariate logistic regression analysis and receiver operating characteristic analysis to the combined data of all patients revealed that the full regression model incorporating all predictor variables, including SOZ and 'MI z-score', best classified the seizure outcome with sensitivity/specificity of 0.86/0.76. The model excluding 'MI z-score' worsened its sensitivity/specificity to 0.86/0.48. Furthermore, the leave-one-out analysis successfully cross-validated the full regression model. Measurement of statistical deviation of MI from the non-epileptic mean on invasive recording is technically feasible. Our analytical technique can be used to evaluate the utility of ECoG biomarkers in epilepsy presurgical evaluation.

Spatiotemporal dynamics of auditory and picture naming-related high-gamma modulations: A study of Japanese-speaking patients.

OBJECTIVE: To characterize the spatiotemporal dynamics of auditory and picture naming-related cortical activation in Japanese-speaking patients. METHODS: Ten patients were assigned auditory naming and picture naming tasks during extraoperative intracranial EEG recording in a tertiary epilepsy center. Time-frequency analysis determined at what electrode sites and at what time windows during each task the amplitude of high-gamma activity (65-95 Hz) was modulated. RESULTS: The superior-temporal gyrus on each hemisphere showed high-gamma augmentation during sentence listening, whereas the left middle-temporal and inferior-frontal gyri showed high-gamma augmentation peaking around stimulus offset. Auditory naming-specific high-gamma augmentation was noted in the bilateral superior-temporal gyri as well as left frontal-parietal-temporal perisylvian network regions, whereas picture naming-specific augmentation was noted in the occipital-fusiform regions, bilaterally. The inferior pre- and postcentral gyri on each hemisphere showed modality-common high-gamma augmentation time-locked to overt responses. CONCLUSIONS: The spatiotemporal dynamics of auditory and picture naming-related high-gamma augmentation in Japanese-speaking patients were qualitatively similar to those previously reported in studies of English-speaking patients. SIGNIFICANCE: The cortical dynamics for auditory sentence recognition are at least partly shared by cohorts speaking two distinct languages. Multicenter studies regarding the clinical utility of high-gamma language mapping across Eastern and Western hemispheres may be feasible.

Four-dimensional functional cortical maps of visual and auditory language: Intracranial recording.

OBJECTIVE: The strength of presurgical language mapping using electrocorticography (ECoG) is its outstanding signal fidelity and temporal resolution, but the weakness includes limited spatial sampling at an individual patient level. By averaging naming-related high-gamma activity at nonepileptic regions across a large number of patients, we provided the functional cortical atlases animating the neural dynamics supporting visual-object and auditory-description naming at the whole brain level. METHODS: We studied 79 patients who underwent extraoperative ECoG recording as epilepsy presurgical evaluation, and generated time-frequency plots and animation videos delineating the dynamics of naming-related high-gamma activity at 70-110 Hz. RESULTS: Naming task performance elicited high-gamma augmentation in domain-specific lower-order sensory areas and inferior-precentral gyri immediately after stimulus onset. High-gamma augmentation subsequently involved widespread neocortical networks with left hemisphere dominance. Left posterior temporal high-gamma augmentation at several hundred milliseconds before response onset exhibited a double dissociation; picture naming elicited high-gamma augmentation preferentially in regions medial to the inferior-temporal gyrus, whereas auditory naming elicited high-gamma augmentation more laterally. The left lateral prefrontal regions including Broca's area initially exhibited high-gamma suppression subsequently followed by high-gamma augmentation at several hundred milliseconds before response onset during both naming tasks. Early high-gamma suppression within Broca's area was more intense during picture compared to auditory naming. Subsequent lateral-prefrontal high-gamma augmentation was more intense during auditory compared to picture naming. SIGNIFICANCE: This study revealed contrasting characteristics in the spatiotemporal dynamics of naming-related neural modulations between tasks. The dynamic atlases of visual and auditory language might be useful for planning of epilepsy surgery. Differential neural activation well explains some of the previously reported observations of domain-specific language impairments following resective epilepsy surgery. Video materials might be beneficial for the education of lay people about how the brain functions differentially during visual and auditory naming.

Phase-amplitude coupling between interictal high-frequency activity and slow waves in epilepsy surgery.

OBJECTIVE: We hypothesized that the modulation index (MI), a summary measure of the strength of phase-amplitude coupling between high-frequency activity (>150 Hz) and the phase of slow waves (3-4 Hz), would serve as a useful interictal biomarker for epilepsy presurgical evaluation. METHODS: We investigated 123 patients who underwent focal cortical resection following extraoperative electrocorticography recording and had at least 1 year of postoperative follow-up. We examined whether consideration of MI would improve the prediction of postoperative seizure outcome. MI was measured at each intracranial electrode site during interictal slow-wave sleep. We compared the accuracy of prediction of patients achieving International League Against Epilepsy class 1 outcome between the full multivariate logistic regression model incorporating MI in addition to conventional clinical, seizure onset zone (SOZ), and neuroimaging variables, and the reduced logistic regression model incorporating all variables other than MI. RESULTS: Ninety patients had class 1 outcome at the time of most recent follow-up (mean follow-up = 5.7 years). The full model had a noteworthy outcome predictive ability, as reflected by regression model fit R2 of 0.409 and area under the curve (AUC) of receiver operating characteristic plot of 0.838. Incomplete resection of SOZ (P < 0.001), larger number of antiepileptic drugs at the time of surgery (P = 0.007), and larger MI in nonresected tissues relative to that in resected tissue (P = 0.020) were independently associated with a reduced probability of class 1 outcome. The reduced model had a lower predictive ability as reflected by R2 of 0.266 and AUC of 0.767. Anatomical variability in MI existed among nonepileptic electrode sites, defined as those unaffected by magnetic resonance imaging lesion, SOZ, or interictal spike discharges. With MI adjusted for anatomical variability, the full model yielded the outcome predictive ability of R2 of 0.422, AUC of 0.844, and sensitivity/specificity of 0.86/0.76. SIGNIFICANCE: MI during interictal recording may provide useful information for the prediction of postoperative seizure outcome.

Metabolic Barriers to T Cell Function in Tumors.

The metabolic programs that drive T cell functions are exquisitely sensitive to cell intrinsic and extrinsic factors, allowing T cells to respond in a fine-tuned manner to a variety of immune challenges and conditions. However, many of the factors essential for effector T cell function are perturbed in the tumor microenvironment, where oncogenic mutations drive unrestrained cancer cell growth that leads to excess nutrient consumption, excess waste excretion, and insufficient oxygen delivery. This imposes metabolic constraints on infiltrating cells that result in dysfunction and loss of potential antitumor activity in both naturally occurring as well as tailored T cells introduced as part of immunotherapy. In this review, we highlight the metabolic properties that characterize tumor-infiltrating T cells, the barriers within the metabolic landscape of the tumor microenvironment, and the opportunities and challenges they present in development of new cancer therapeutics.

Four-dimensional map of the human early visual system.

OBJECTIVE: We generated a large-scale, four-dimensional map of neuronal modulations elicited by full-field flash stimulation. METHODS: We analyzed electrocorticography (ECoG) recordings from 63 patients with focal epilepsy, and delineated the spatial-temporal dynamics of visually-elicited high-gamma70-110 Hz amplitudes on a standard brain template. We then clarified the neuronal events underlying visual evoked potential (VEP) components, by correlating with high-gamma amplitude measures. RESULTS: The medial-occipital cortex initially revealed rapid neural activation followed by prolonged suppression, reflected by augmentation of high-gamma activity lasting up to 100 ms followed by attenuation lasting up to 1000 ms, respectively. With a number of covariate factors incorporated into a prediction model, the eccentricity representation independently predicted the magnitude of post-activation suppression, which was more intense in regions representing more parafoveal visual fields compared to those of more peripheral fields. The initial negative component on VEP was sharply contoured and co-occurred with early high-gamma augmentation, whose offset then co-occurred with a large positive VEP peak. A delayed negative VEP peak was blunt and co-occurred with prolonged high-gamma attenuation. CONCLUSIONS: Eccentricity-dependent gradient in neural suppression in the medial-occipital region may explain the functional difference between peripheral and parafoveal/central vision. Early negative and positive VEP components may reflect neural activation, whereas a delayed negative VEP peak reflecting neural suppression. SIGNIFICANCE: Our observation provides the mechanistic rationale for transient scotoma or mild flash-blindness, characterized by physiological afterimage preferentially formed in central vision following intense but non-injurious light exposure.

Therapeutic potential of DNA methyltransferase inhibitors with immune checkpoint inhibitor therapy in breast cancer.

However, ICI therapy has thus far demonstrated limited efficacy in breast cancers, where tumor mutation rates are intermediate. Nonetheless, because of limited but positive signals in early trials, combinations of therapies to enhance anti-tumor immunity, and thus response to ICIs in breast cancer, are actively being sought. Our laboratory recently found that guadecitabine, a next-generation DNA methyltransferase inhibitor (DMTi), potentiated cytotoxic CD8+ T cell responses in breast cancer, which appeared to occur by the following mechanisms: (1) DMTi treatment hypomethylated and up-regulated both baseline and IFN-γ-induced MHC-I expression, thereby enhancing antigen presentation capacity, (2) DMTi treatment increased Cxcr3 ligands/chemokines (i.e., Cxcl9, Cxcl10, and Cxcl11) expression and recruited cytotoxic CD8+ T cells into the tumors and (3) DMTi treatment activated NFκB signaling, presumably through the expression of endogenous retroviral (ERV) sequences in tumor cells, initiating an innate response observed in other solid tumor types [Luo et al., Nat Commun 9(1):248]. Most importantly, DMTi treatment primed breast cancer and improved responses to anti-PD-L1 therapy.