LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

Vascular physiology drives functional brain networks.

We present the first evidence for vascular regulation driving fMRI signals in specific functional brain networks. Using concurrent neuronal and vascular stimuli, we collected 30 BOLD fMRI datasets in 10 healthy individuals: a working memory task, flashing checkerboard stimulus, and CO2 inhalation challenge were delivered in concurrent but orthogonal paradigms. The resulting imaging data were averaged together and decomposed using independent component analysis, and three "neuronal networks" were identified as demonstrating maximum temporal correlation with the neuronal stimulus paradigms: Default Mode Network, Task Positive Network, and Visual Network. For each of these, we observed a second network component with high spatial overlap. Using dual regression in the original 30 datasets, we extracted the time-series associated with these network pairs and calculated the percent of variance explained by the neuronal or vascular stimuli using a normalized R2 parameter. In each pairing, one network was dominated by the appropriate neuronal stimulus, and the other was dominated by the vascular stimulus as represented by the end-tidal CO2 time-series recorded in each scan. We acquired a second dataset in 8 of the original participants, where no CO2 challenge was delivered and CO2 levels fluctuated naturally with breathing variations. Although splitting of functional networks was not robust in these data, performing dual regression with the network maps from the original analysis in this new dataset successfully replicated our observations. Thus, in addition to responding to localized metabolic changes, the brain's vasculature may be regulated in a coordinated manner that mimics (and potentially supports) specific functional brain networks. Multi-modal imaging and advances in fMRI acquisition and analysis could facilitate further study of the dual nature of functional brain networks. It will be critical to understand network-specific vascular function, and the behavior of a coupled vascular-neural network, in future studies of brain pathology.

Multi-site harmonization of 7 tesla MRI neuroimaging protocols.

Increasing numbers of 7 T (7 T) magnetic resonance imaging (MRI) scanners are in research and clinical use. 7 T MRI can increase the scanning speed, spatial resolution and contrast-to-noise-ratio of many neuroimaging protocols, but technical challenges in implementation have been addressed in a variety of ways across sites. In order to facilitate multi-centre studies and ensure consistency of findings across sites, it is desirable that 7 T MRI sites implement common high-quality neuroimaging protocols that can accommodate different scanner models and software versions. With the installation of several new 7 T MRI scanners in the United Kingdom, the UK7T Network was established with an aim to create a set of harmonized structural and functional neuroimaging sequences and protocols. The Network currently includes five sites, which use three different scanner platforms, provided by two different vendors. Here we describe the harmonization of functional and anatomical imaging protocols across the three different scanner models, detailing the necessary changes to pulse sequences and reconstruction methods. The harmonized sequences are fully described, along with implementation details. Example datasets acquired from the same subject on all Network scanners are made available. Based on these data, an evaluation of the harmonization is provided. In addition, the implementation and validation of a common system calibration process is described.

Multi-centre, multi-vendor reproducibility of 7T QSM and R2* in the human brain: Results from the UK7T study.

INTRODUCTION: We present the reliability of ultra-high field T2* MRI at 7T, as part of the UK7T Network's "Travelling Heads" study. T2*-weighted MRI images can be processed to produce quantitative susceptibility maps (QSM) and R2* maps. These reflect iron and myelin concentrations, which are altered in many pathophysiological processes. The relaxation parameters of human brain tissue are such that R2* mapping and QSM show particularly strong gains in contrast-to-noise ratio at ultra-high field (7T) vs clinical field strengths (1.5-3T). We aimed to determine the inter-subject and inter-site reproducibility of QSM and R2* mapping at 7T, in readiness for future multi-site clinical studies. METHODS: Ten healthy volunteers were scanned with harmonised single- and multi-echo T2*-weighted gradient echo pulse sequences. Participants were scanned five times at each "home" site and once at each of four other sites. The five sites had 1× Philips, 2× Siemens Magnetom, and 2× Siemens Terra scanners. QSM and R2* maps were computed with the Multi-Scale Dipole Inversion (MSDI) algorithm (https://github.com/fil-physics/Publication-Code). Results were assessed in relevant subcortical and cortical regions of interest (ROIs) defined manually or by the MNI152 standard space. RESULTS AND DISCUSSION: Mean susceptibility (χ) and R2* values agreed broadly with literature values in all ROIs. The inter-site within-subject standard deviation was 0.001-0.005 ppm (χ) and 0.0005-0.001 ms-1 (R2*). For χ this is 2.1-4.8 fold better than 3T reports, and 1.1-3.4 fold better for R2*. The median ICC from within- and cross-site R2* data was 0.98 and 0.91, respectively. Multi-echo QSM had greater variability vs single-echo QSM especially in areas with large B0 inhomogeneity such as the inferior frontal cortex. Across sites, R2* values were more consistent than QSM in subcortical structures due to differences in B0-shimming. On a between-subject level, our measured χ and R2* cross-site variance is comparable to within-site variance in the literature, suggesting that it is reasonable to pool data across sites using our harmonised protocol. CONCLUSION: The harmonized UK7T protocol and pipeline delivers on average a 3-fold improvement in the coefficient of reproducibility for QSM and R2* at 7T compared to previous reports of multi-site reproducibility at 3T. These protocols are ready for use in multi-site clinical studies at 7T.

Venous contribution to sodium MRI in the human brain.

PURPOSE: Sodium MRI shows great promise as a marker for cerebral metabolic dysfunction in stroke, brain tumor, and neurodegenerative pathologies. However, cerebral blood vessels, whose volume and function are perturbed in these pathologies, have elevated sodium concentrations relative to surrounding tissue. This study aims to assess whether this fluid compartment could bias measurements of tissue sodium using MRI. METHODS: Density-weighted and B1 corrected sodium MRI of the brain was acquired in 9 healthy participants at 4.7T. Veins were identified using co-registered 1 H T2∗ -weighted images and venous partial volume estimates were calculated by down-sampling the finer spatial resolution venous maps from the T2∗ -weighted images to the coarser spatial resolution of the sodium data. Linear regressions of venous partial volume estimates and sodium signal were performed for regions of interest including just gray matter, just white matter, and all brain tissue. RESULTS: Linear regression demonstrated a significant venous sodium contribution above the underlying tissue signal. The apparent venous sodium concentrations derived from regression were 65.8 ± 4.5 mM (all brain tissue), 71.0 ± 7.4 mM (gray matter), and 55.0 ± 4.7 mM (white matter). CONCLUSION: Although the partial vein linear regression did not yield the expected sodium concentration in blood (~87 mM), likely the result of point spread function smearing, this regression highlights that blood compartments may bias brain tissue sodium signals across neurological conditions where blood volumes may differ.

Whole brain 31 P MRSI at 7T with a dual-tuned receive array.

PURPOSE: The design and performance of a novel head coil setup for 31 P spectroscopy at ultra-high field strengths (7T) is presented. The described system supports measurements at both the 1 H and 31 P resonance frequencies. METHODS: The novel coil consists of 2, actively detunable, coaxial birdcage coils to give homogeneous transmit, combined with a double resonant 30 channel receive array. This allows for anatomical imaging combined with 31 P acquisitions over the whole head, without changing coils or disturbing the subject. A phosphate buffer phantom and 3 healthy volunteers were scanned with a pulse acquire CSI sequence using both the novel array coil and a conventional transceiver birdcage. Four different methods of combining the array channels were compared at 3 different levels of SNR. RESULTS: The novel coil setup delivers significantly increased 31 P SNR in the peripheral regions of the brain, reaching up to factor 8, while maintaining comparable performance relative to the birdcage in the center. CONCLUSIONS: The new system offers the potential to acquire whole brain 31 P MRSI with superior signal relative to the standard options.

Changes in arterial cerebral blood volume during lower body negative pressure measured with MRI.

Cerebral Autoregulation (CA), defined as the ability of the cerebral vasculature to maintain stable levels of blood flow despite changes in systemic blood pressure, is a critical factor in neurophysiological health. Magnetic resonance imaging (MRI) is a powerful technique for investigating cerebrovascular function, offering high spatial resolution and wide fields of view (FOV), yet it is relatively underutilized as a tool for assessment of CA. The aim of this study was to demonstrate the potential of using MRI to measure changes in cerebrovascular resistance in response to lower body negative pressure (LBNP). A Pulsed Arterial Spin Labeling (PASL) approach with short inversion times (TI) was used to estimate cerebral arterial blood volume (CBVa) in eight healthy subjects at baseline and -40mmHg LBNP. We estimated group mean CBVa values of 3.13 ± 1.00 and 2.70 ± 0.38 for baseline and lbnp respectively, which were the result of a differential change in CBVa during -40mmHg LBNP that was dependent on baseline CBVa. These data suggest that the PASL CBVa estimates are sensitive to the complex cerebrovascular response that occurs during the moderate orthostatic challenge delivered by LBNP, which we speculatively propose may involve differential changes in vascular tone within different segments of the arterial vasculature. These novel data provide invaluable insight into the mechanisms that regulate perfusion of the brain, and establishes the use of MRI as a tool for studying CA in more detail.

Coupling between cerebral blood flow and cerebral blood volume: Contributions of different vascular compartments.

A better understanding of the coupling between changes in cerebral blood flow (CBF) and cerebral blood volume (CBV) is vital for furthering our understanding of the BOLD response. The aim of this study was to measure CBF-CBV coupling in different vascular compartments during neural activation. Three haemodynamic parameters were measured during a visual stimulus. Look-Locker flow-sensitive alternating inversion recovery was used to measure changes in CBF and arterial CBV (CBVa ) using sequence parameters optimized for each contrast. Changes in total CBV (CBVtot ) were measured using a gadolinium-based contrast agent technique. Haemodynamic changes were extracted from a region of interest based on voxels that were activated in the CBF experiments. The CBF-CBVtot coupling constant αtot was measured as 0.16 ± 0.14 and the CBF-CBVa coupling constant αa was measured as 0.65 ± 0.24. Using a two-compartment model of the vasculature (arterial and venous), the change in venous CBV (CBVv ) was predicted for an assumed value of baseline arterial and venous blood volume. These results will enhance the accuracy and reliability of applications that rely on models of the BOLD response, such as calibrated BOLD.

Arterial CO2 Fluctuations Modulate Neuronal Rhythmicity: Implications for MEG and fMRI Studies of Resting-State Networks.

UNLABELLED: A fast emerging technique for studying human resting state networks (RSNs) is based on spontaneous temporal fluctuations in neuronal oscillatory power, as measured by magnetoencephalography. However, it has been demonstrated recently that this power is sensitive to modulations in arterial CO2 concentration. Arterial CO2 can be modulated by natural fluctuations in breathing pattern, as might typically occur during the acquisition of an RSN experiment. Here, we demonstrate for the first time the fine-scale dependence of neuronal oscillatory power on arterial CO2 concentration, showing that reductions in alpha, beta, and gamma power are observed with even very mild levels of hypercapnia (increased arterial CO2). We use a graded hypercapnia paradigm and participant feedback to rule out a sensory cause, suggesting a predominantly physiological origin. Furthermore, we demonstrate that natural fluctuations in arterial CO2, without administration of inspired CO2, are of a sufficient level to influence neuronal oscillatory power significantly in the delta-, alpha-, beta-, and gamma-frequency bands. A more thorough understanding of the relationship between physiological factors and cortical rhythmicity is required. In light of these findings, existing results, paradigms, and analysis techniques for the study of resting-state brain data should be revisited. SIGNIFICANCE STATEMENT: In this study, we show for the first time that neuronal oscillatory power is intimately linked to arterial CO2 concentration down to the fine-scale modulations that occur during spontaneous breathing. We extend these results to demonstrate a correlation between neuronal oscillatory power and spontaneous arterial CO2 fluctuations in awake humans at rest. This work identifies a need for studies investigating resting-state networks in the human brain to measure and account for the impact of spontaneous changes in arterial CO2 on the neuronal signals of interest. Changes in breathing pattern that are time locked to task performance could also lead to confounding effects on neuronal oscillatory power when considering the electrophysiological response to functional stimulation.

Field strength dependence of grey matter R2* on venous oxygenation.

The relationship between venous blood oxygenation and change in transverse relaxation rate (ΔR2*) plays a key role in calibrated BOLD fMRI. This relationship, defined by the parameter ß, has previously been determined using theoretical simulations and experimental measures. However, these earlier studies have been confounded by the change in venous cerebral blood volume (CBV) in response to functional tasks. This study used a double-echo gradient echo EPI scheme in conjunction with a graded isocapnic hyperoxic sequence to assess quantitatively the relationship between the fractional venous blood oxygenation (1-Yv) and transverse relaxation rate of grey matter (ΔR2⁢GM*), without inducing a change in vCBV. The results demonstrate that the relationship between ΔR2* and fractional venous oxygenation at all magnet field strengths studied was adequately described by a linear relationship. The gradient of this relationship did not increase monotonically with field strength, which may be attributed to the relative contributions of intravascular and extravascular signals which will vary with both field strength and blood oxygenation.

Specification of regional intestinal stem cell identity during Drosophila metamorphosis.

In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR). BMP signaling is also involved in the modulation of intestinal stem cell (ISC) proliferation in response to injury. How ISCs are able to respond to the same signaling pathway in a regionally different manner is currently unknown. Here, we show that dual use of the BMP signaling pathway in the midgut is possible because BMP signals are only capable of transforming ISC and enterocyte identity during a defined window of metamorphosis. ISC heterogeneity is established prior to adulthood and then maintained in cooperation with regional signals from surrounding tissue. Our data provide a conceptual framework for how other tissues maintained by regional stem cells might be patterned and establishes the pupal and adult midgut as a novel genetic platform for identifying genes necessary for regional stem cell specification and maintenance.

The absolute CBF response to activation is preserved during elevated perfusion: Implications for neurovascular coupling measures.

Functional magnetic resonance imaging (fMRI) techniques in which the blood oxygenation level dependent (BOLD) and cerebral blood flow (CBF) response to a neural stimulus are measured, can be used to estimate the fractional increase in the cerebral metabolic rate of oxygen consumption (CMRO2) that accompanies evoked neural activity. A measure of neurovascular coupling is obtained from the ratio of fractional CBF and CMRO2 responses, defined as n, with the implicit assumption that relative rather than absolute changes in CBF and CMRO2 adequately characterise the flow-metabolism response to neural activity. The coupling parameter n is important in terms of its effect on the BOLD response, and as potential insight into the flow-metabolism relationship in both normal and pathological brain function. In 10 healthy human subjects, BOLD and CBF responses were measured to test the effect of baseline perfusion (modulated by a hypercapnia challenge) on the coupling parameter n during graded visual stimulation. A dual-echo pulsed arterial spin labelling (PASL) sequence provided absolute quantification of CBF in baseline and active states as well as relative BOLD signal changes, which were used to estimate CMRO2 responses to the graded visual stimulus. The absolute CBF response to the visual stimuli were constant across different baseline CBF levels, meaning the fractional CBF responses were reduced at the hyperperfused baseline state. For the graded visual stimuli, values of n were significantly reduced during hypercapnia induced hyperperfusion. Assuming the evoked neural responses to the visual stimuli are the same for both baseline CBF states, this result has implications for fMRI studies that aim to measure neurovascular coupling using relative changes in CBF. The coupling parameter n is sensitive to baseline CBF, which would confound its interpretation in fMRI studies where there may be significant differences in baseline perfusion between groups. The absolute change in CBF, as opposed to the change relative to baseline, may more closely match the underlying increase in neural activity in response to a stimulus.

The effect of isocapnic hyperoxia on neurophysiology as measured with MRI and MEG.

The physiological effect of hyperoxia has been poorly characterize d, with studies reporting conflicting results on the role of hyperoxia as a vasoconstrictor. It is not clear whether hyperoxia is the primary contributor to vasoconstriction or whether induced changes in CO2 that commonly accompany hyperoxia are a factor. As calibrated BOLD fMRI based on hyperoxia becomes more widely used, it is essential to understand the effects of oxygen on resting cerebral physiology. This study used a RespirAct™ system to deliver a repeatable isocapnic hyperoxia stimulus to investigate the independent effect of O2 on cerebral physiology, removing any potential confounds related to altered CO2. T1-independent Phase Contrast MRI was used to demonstrate that isocapnic hyperoxia has no significant effect on carotid blood flow (normoxia 201 ± 11 ml/min, -0.3% ± 0.8% change during hyperoxia, p = 0.8), while Look Locker ASL was used to demonstrate that there is no significant change in arterial cerebral blood volume (normoxia 1.3% ± 0.4%, -0.5 ± 5% change during hyperoxia). These are in contrast to significant changes in carotid blood flow observed for hypercapnia (6.8% ± 1.5%/mm Hg CO2). In addition, magnetoencephalography provided a method to monitor the effect of isocapnic hyperoxia on neuronal oscillatory power. In response to hyperoxia, a significant focal decrease in oscillatory power was observed across the alpha, beta and low gamma bands in the occipital lobe, compared to a more global significant decrease on hypercapnia. This work suggests that isocapnic hyperoxia provides a more reliable stimulus than hypercapnia for calibrated BOLD, and that previous reports of vasoconstriction during hyperoxia probably reflect the effects of hyperoxia-induced changes in CO2. However, hyperoxia does induce changes in oscillatory power consistent with an increase in vigilance, but these changes are smaller than those observed under hypercapnia. The effect of this change in neural activity on calibrated BOLD using hyperoxia or combined hyperoxia and hypercapnia needs further investigation.

Hemispheric asymmetry in cerebrovascular reactivity of the human primary motor cortex: an in vivo study at 7 T.

Current functional MRI (fMRI) approaches assess underlying neuronal activity through monitoring the related local variations in cerebral blood oxygenation, blood volume and blood flow. This vascular response is likely to vary across brain regions and across individuals, depending on the composition of the local vascular bed and on the vascular capacity to dilate. The most widely used technique uses the blood oxygen level dependent (BOLD) fMRI signal, which arises from a complex combination of all of these factors. The model of handedness provides a case where one brain region (dominant motor cortex) is known to have a stronger BOLD response over another (non-dominant motor cortex) during hand motor task performance. We predict that this is accompanied by a higher vascular reactivity in the dominant motor cortex, when compared with the non-dominant motor cortex. Precise measurement of end-tidal CO2 and a novel sinusoidal CO2 respiratory challenge were combined with the high sensitivity and finer spatial resolution available for fMRI at 7 T to measure BOLD cerebrovascular reactivity (CVR) in eight healthy male participants. BOLD CVR was compared between the left (dominant) and right (non-dominant) primary motor cortices of right-handed adults. Hemispheric asymmetry in vascular reactivity was predicted and observed in the primary motor cortex (left CVR = 0.60 ± 0.15%/mm Hg; right CVR = 0.47 ± 0.08%/mm Hg; left CVR > right CVR, P = 0.04), the first reported evidence of such a vascular difference. These findings demonstrate a cerebral vascular asymmetry between the left and right primary motor cortex. The origin of this asymmetry largely arises from the contribution of large draining veins. This work has implications for future motor laterality studies that use BOLD, and it is also suggestive of a vascular plasticity in the human primary motor cortex.

Global intravascular and local hyperoxia contrast phase-based blood oxygenation measurements.

The measurement of venous cerebral blood oxygenation (Yv) has potential applications in the study of patient groups where oxygen extraction and/or metabolism are compromised. It is also useful for fMRI studies to assess the stimulus-induced changes in Yv, particularly since basal Yv partially accounts for inter-subject variation in the haemodynamic response to a stimulus. A range of MRI-based methods of measuring Yv have been developed recently. Here, we use a method based on the change in phase in the MR image arising from the field perturbation caused by deoxygenated haemoglobin in veins. We build on the existing phase based approach (Method I), where Yv is measured in a large vein (such as the superior sagittal sinus) based on the field shift inside the vein with assumptions as to the vein's shape and orientation. We demonstrate two novel modifications which address limitations of this method. The first modification (Method II), maps the actual form of the vein, rather than assume a given shape and orientation. The second modification (Method III) uses the intra and perivascular phase change in response to a known change in Yv on hyperoxia to measure normoxic Yv in smaller veins. Method III can be applied to veins whose shape, size and orientation are not accurately known, thus allowing more localised measures of venous oxygenation. Results demonstrate that the use of an overly fine spatial filter caused an overestimation in Yv for Method I, whilst the measurement of Yv using Method II was less sensitive to this bias, giving Yv = 0.62 ± 0.03. Method III was applied to mapping of Yv in local veins across the brain, yielding a distribution of values with a mode of Yv = 0.661 ± 0.008.

In search of a marker of altered cerebrovascular function in hypertension: Analysis of the fractional amplitude of low-frequency fluctuations in UK Biobank resting state fMRI data.

The selfish brain mechanism proposes that in some patients with impaired cerebral blood flow (CBF) or cerebrovascular function, hypertension may develop as a compensatory mechanism that aims to maintain CBF by increasing systemic blood pressure through an increase in cardiovascular sympathetic tone. The amplitude of low frequency fluctuations (ALFF) in the resting state blood oxygenation level dependent (BOLD) functional MRI signal has been previously posited as an index of cerebrovascular reactivity. We investigated whether regional fractional ALFF (fALFF) differs between 2054 hypertensives and 1724 normotensives using data from the UK Biobank dataset. Our primary hypothesis was that cerebrovascular function in the medulla and other regions involved in sympathetic regulation differs between hypertensives and normotensives, and that this is reflected by regional variations in fALFF. There is a significant regional variation in fALFF (F(14) =1126.17, p < 2 × 10-16, partial η2 = 0.22), but this regional variation does not differ between hypertensives and normotensives (F(14) = 0.23, p = 0.99, partial η2 = 8 × 10-5). Prospective longitudinal studies of cerebral haemodynamics in hypertensives and normotensives are required to further investigate the selfish brain mechanism.

Assessment of brainstem function and haemodynamics by MRI: challenges and clinical prospects.

MRI offers techniques for non-invasively measuring a range of aspects of brain tissue function. Blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) is widely used to assess neural activity, based on the brain's haemodynamic response, while arterial spin labelling (ASL) MRI is a non-invasive method of quantitatively mapping cerebral perfusion. Both techniques can be applied to measure cerebrovascular reactivity (CVR), an important marker of the health of the cerebrovascular system. BOLD, ASL and CVR have been applied to study a variety of disease processes and are already used in certain clinical circumstances. The brainstem is a critical component of the central nervous system and is implicated in a variety of disease processes. However, its function is difficult to study using MRI because of its small size and susceptibility to physiological noise. In this article, we review the physical and biological underpinnings of BOLD and ASL and their application to measure CVR, discuss the challenges associated with applying them to the brainstem and the opportunities for brainstem MRI in the research and clinical settings. With further optimisation, functional MRI techniques could feasibly be used to assess brainstem haemodynamics and neural activity in the clinical setting.