Maintenance of CD4 T cell fitness through regulation of Foxo1.

Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor ß-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.

Cutting Edge: Dynamic Expression of Id3 Defines the Stepwise Differentiation of Tissue-Resident Regulatory T Cells.

Foxp3+ regulatory T (TR) cells are phenotypically and functionally diverse and broadly distributed in lymphoid and nonlymphoid tissues. However, the pathways guiding the differentiation of tissue-resident TR cell populations have not been well defined. By regulating E-protein function, Id3 controls the differentiation of CD8+ effector T cells and is essential for TR cell maintenance and function. We show that dynamic expression of Id3 helps define three distinct mouse TR cell populations: Id3+CD62LhiCD44lo central TR cells, Id3+CD62LloCD44hi effector TR (eTR) cells, and Id3- eTR cells. Adoptive transfer experiments and transcriptome analyses support a stepwise model of differentiation from Id3+ central TR to Id3+ eTR to Id3- eTR cells. Furthermore, Id3- eTR cells have high expression of functional inhibitory markers and a transcriptional signature of tissue-resident TR cells. Accordingly, Id3- eTR cells are highly enriched in nonlymphoid organs but virtually absent from blood and lymph. Thus, we propose that tissue-resident TR cells develop in a multistep process associated with Id3 downregulation.

Convective forces increase rostral delivery of intrathecal radiotracers and antisense oligonucleotides in the cynomolgus monkey nervous system.

BACKGROUND: The intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys. METHODS: There are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99mTc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging. RESULTS: It was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm3. CONCLUSIONS: These results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.

Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation.

Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögren's syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.

The perivascular pathways for influx of cerebrospinal fluid are most efficient in the midbrain.

Although there are no conventional lymphatic vessels in the brain, fluid and solutes drain along basement membranes (BMs) of cerebral capillaries and arteries towards the subarachnoid space and cervical lymph nodes. Convective influx/glymphatic entry of the cerebrospinal fluid (CSF) into the brain parenchyma occurs along the pial-glial BMs of arteries. This project tested the hypotheses that pial-glial BM of arteries are thicker in the midbrain, allowing more glymphatic entry of CSF. The in vivo MRI and PET images were obtained from a 4.2-year-old dog, whereas the post-mortem electron microscopy was performed in a 12-year-old dog. We demonstrated a significant increase in the thickness of the pial-glial BM in the midbrain compared with the same BM in different regions of the brain and an increase in the convective influx of fluid from the subarachnoid space. These results are highly significant for the intrathecal drug delivery into the brain, indicating that the midbrain is better equipped for convective influx/glymphatic entry of the CSF.

B-cell intrinsic TLR7 signals promote depletion of the marginal zone in a murine model of Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) exhibit prominent defects in splenic marginal zone (MZ), resulting in abnormal T-cell-independent antibody responses and increased bacterial infections. B-cell-intrinsic deletion of the affected gene WAS protein (WASp) markedly reduces splenic MZ B cells, without impacting the rate of MZ B-cell development, suggesting that abnormal B-cell retention within the MZ accounts for MZ defects in WAS. Since WASp regulates integrin-dependent actin cytoskeletal rearrangement, we previously hypothesized that defective B-cell integrin function promotes MZ depletion. In contrast, we now report that B-cell-intrinsic deletion of the TLR signaling adaptor MyD88 is sufficient to restore the MZ in WAS. We further identify TLR7, an endosomal single-stranded RNA (ssRNA) receptor, as the MyD88-dependent receptor responsible for WAS MZ depletion. These findings implicate spontaneous activation of MZ B cells by ssRNA-containing self-ligands (likely derived from circulating apoptotic material) as the mechanism underlying MZ depletion in WAS. Together, these data suggest a previously unappreciated role for B-cell intrinsic TLR signals in MZ homeostasis, of relevance to both pathogen responses and to the development of systemic autoimmunity.

Sex differences in the brain's dopamine signature of cigarette smoking.

Cigarette smoking is a major public health danger. Women and men smoke for different reasons and cessation treatments, such as the nicotine patch, are preferentially beneficial to men. The biological substrates of these sex differences are unknown. Earlier PET studies reported conflicting findings but were each hampered by experimental and/or analytical limitations. Our new image analysis technique, lp-ntPET (Normandin et al., 2012; Morris et al., 2013; Kim et al., 2014), has been optimized for capturing brief (lasting only minutes) and highly localized dopaminergic events in dynamic PET data. We coupled our analysis technique with high-resolution brain scanning and high-frequency motion correction to create the optimal experiment for capturing and characterizing the effects of smoking on the mesolimbic dopamine system in humans. Our main finding is that male smokers smoking in the PET scanner activate dopamine in the right ventral striatum during smoking but female smokers do not. This finding-men activating more ventrally than women-is consistent with the established notion that men smoke for the reinforcing drug effect of cigarettes whereas women smoke for other reasons, such as mood regulation and cue reactivity. lp-ntPET analysis produces a novel multidimensional endpoint: voxel-level temporal patterns of neurotransmitter release ("DA movies") in individual subjects. By examining these endpoints quantitatively, we demonstrate that the timing of dopaminergic responses to cigarette smoking differs between men and women. Men respond consistently and rapidly in the ventral striatum whereas women respond faster in a discrete subregion of the dorsal putamen.

A T cell extrinsic mechanism by which IL-2 dampens Th17 differentiation.

Genetic variants in il2 and il2ra have been associated with autoimmune disease susceptibility in both genome-wide association studies (GWAS) in humans and in genetic linkage studies in experimental models of autoimmunity. Specifically, genetic variants resulting in a low IL-2 phenotype are susceptibility alleles while variants resulting in a high IL-2 phenotype are resistance alleles. The association of high IL-2 phenotypes with resistance has been attributed primarily to the T cell intrinsic promotion of regulatory T cell development, maintenance, and function; however, IL-2 can also act T cell intrinsically to dampen differentiation of pathogenic IL-17-producing Th17 cells. Here, we have uncovered a novel T cell extrinsic mechanism whereby IL-2 promotes both IFN-γ and IL-27 production from tissue resident macrophages which in turn dampen the differentiation of pathogenic Th17 cells.

Test-retest reproducibility of the metabotropic glutamate receptor 5 ligand [¹8F]FPEB with bolus plus constant infusion in humans.

PURPOSE: [(18)F]FPEB is a promising PET radioligand for the metabotropic glutamate receptor 5 (mGluR5), a potential target for the treatment of neuropsychiatric diseases. The purpose of this study was to evaluate the test-retest reproducibility of [(18)F]FPEB in the human brain. METHODS: Seven healthy male subjects were scanned twice, 3 - 11 weeks apart. Dynamic data were acquired using bolus plus infusion of 162 ± 32 MBq [(18)F]FPEB. Four methods were used to estimate volume of distribution (V T): equilibrium analysis (EQ) using arterial (EQA) or venous input data (EQV), MA1, and a two-tissue compartment model (2 T). Binding potential (BP ND) was also estimated using cerebellar white matter (CWM) or gray matter (CGM) as the reference region using EQ, 2 T and MA1. Absolute test-retest variability (aTRV) of V T and BP ND were calculated for each method. Venous blood measurements (C V) were compared with arterial input (C A) to examine their usability in EQ analysis. RESULTS: Regional V T estimated by the four methods displayed a high degree of agreement (r (2) ranging from 0.83 to 0.99 among the methods), although EQA and EQV overestimated V T by a mean of 9 % and 7 %, respectively, compared to 2 T. Mean values of aTRV of V T were 11 % by EQA, 12 % by EQV, 14 % by MA1 and 14 % by 2 T. Regional BP ND also agreed well among the methods and mean aTRV of BP ND was 8 - 12 % (CWM) and 7 - 9 % (CGM). Venous and arterial blood concentrations of [(18)F]FPEB were well matched during equilibrium (C V = 1.01 · C A, r (2) = 0.95). CONCLUSION: [(18)F]FPEB binding shows good TRV with minor differences among analysis methods. Venous blood can be used as an alternative for input function measurement instead of arterial blood in EQ analysis. Thus, [(18)F]FPEB is an excellent PET imaging tracer for mGluR5 in humans.

Voxelwise lp-ntPET for detecting localized, transient dopamine release of unknown timing: sensitivity analysis and application to cigarette smoking in the PET scanner.

The "linear parametric neurotransmitter PET" (lp-ntPET) model estimates time variation in endogenous neurotransmitter levels from dynamic PET data. The pattern of dopamine (DA) change over time may be an important element of the brain's response to addictive substances such as cigarettes or alcohol. We have extended the lp-ntPET model from the original region of interest (ROI) - based implementation to be able to apply the model at the voxel level. The resulting endpoint is a dynamic image, or movie, of transient neurotransmitter changes. Simulations were performed to select threshold values to reduce the false positive rate when applied to real (11)C-raclopride PET data. We tested the new voxelwise method on simulated data, and finally, we applied it to (11)C-raclopride PET data of subjects smoking cigarettes in the PET scanner. In simulation, the temporal precision of neurotransmitter response was shown to be similar to that of ROI-based lp-ntPET (standard deviation ∼ 3 min). False positive rates for the voxelwise method were well controlled by combining a statistical threshold (the F-test) with a new spatial (cluster-size) thresholding operation. Sensitivity of detection for the new algorithm was greater than 80% for the case of short-lived DA changes that occur in subregions of the striatum as might be the case with cigarette smoking. Finally, in (11)C-raclopride PET data, DA movies reveal for the first time that different temporal patterns of the DA response to smoking may exist in different subregions of the striatum. These spatiotemporal patterns of neurotransmitter change created by voxelwise lp-ntPET may serve as novel biomarkers for addiction and/or treatment efficacy.

A transgenic model of central nervous system autoimmunity mediated by CD4+ and CD8+ T and B cells.

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and ß-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.

mWTX-330, an IL-12 INDUKINE Molecule, Activates and Reshapes Tumor-Infiltrating CD8+ T and NK Cells to Generate Antitumor Immunity.

IL-12 is a pleotropic inflammatory cytokine, which has broad stimulatory effects on various immune cell populations, making it an attractive target for cancer immunotherapy. However, despite generating robust antitumor activity in syngeneic murine tumor models, clinical administration of IL-12 has been limited by severe toxicity. mWTX-330 is a selectively inducible INDUKINE molecule comprised of a half-life extension domain and an inactivation domain linked to chimeric IL-12 by tumor protease-sensitive linkers. Systemic administration of mWTX-330 in mice was well tolerated, resulted in robust antitumor immunity in multiple tumor models, and preferentially activated tumor-infiltrating immune cells rather than immune cells present in peripheral tissues. Antitumor activity was dependent on in vivo processing of the protease cleavable linkers and required CD8+ T cells for full efficacy. Within the tumor, mWTX-330 increased the frequency of cross-presenting dendritic cells (DC), activated natural killer (NK) cells, skewed conventional CD4+ T cells toward a T helper 1 (TH1) phenotype, drove regulatory T cells (Treg) fragility, and increased the frequency of polyfunctional CD8+ T cells. mWTX-330 treatment also increased the clonality of tumor-infiltrating T cells by expanding underrepresented T-cell receptor (TCR) clones, drove CD8+ T and NK cells towards increased mitochondrial respiration and fitness, and decreased the frequency of TOX+ exhausted CD8+ T cells within the tumor. A fully human version of this INDUKINE molecule was stable in human serum, was reliably and selectively processed by human tumor samples, and is currently in clinical development.

Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression.

IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment. See related Spotlight by Silva, p. 544.

Imaging of alcohol-induced dopamine release in rats:preliminary findings with [(11) C]raclopride PET.

Microdialysis studies report that systemic alcohol increases extracellular dopamine (DA) in the rat striatum. The present study examined whether changes in striatal DA could be detected in rats using small animal positron emission tomography (PET). PET images were acquired in 44 alcohol-naïve male Wistar and alcohol-preferring (P) rats. Subjects received up to three [(11) C]raclopride scans (rest, alcohol, and saline). Animals were anesthetized with isoflurane and secured on a stereotactic-like holder during all scans. Blood samples were collected from the tail or lateral saphenous vein of 12 animals 10 min after tracer injection for determination of blood alcohol concentration (BAC). Time activity curves were extracted from the striatum and the cerebellum and binding potential (BP(ND) ) was calculated as a measure of D(2) receptor availability. Wistars given 1.0 g kg(-1) alcohol (20%v/v) i.v. or 3.0 g kg(-1) alcohol (20%v/v) i.p. showed significant alcohol-induced decreases in BP(ND) . In P rats (given 1.5, 2.25, or 3.0 g kg(-1) alcohol), no individual group showed a statistical effect of alcohol on BP(ND) , but taken together, all P rats receiving i.p. alcohol had significantly lower BP(ND) than rest or saline scans. Large decreases in BP(ND) were primarily observed in rats with BAC above 200 mg%. Also, a significant difference was found between baseline BP(ND) of Wistars who had undergone jugular catheterization surgery for i.v. alcohol administration and those who had not. Preliminary results suggest that alcohol-induced DA release in the rat striatum is detectable using small animal PET given sufficiently large cohorts and adequate blood alcohol levels.

In Vivo Expansion of Antigen-Specific Regulatory T Cells through Staggered Fc.IL-2 Mutein Dosing and Antigen-Specific Immunotherapy.

In mice, Ag administration in the absence of adjuvant typically elicits tolerogenic immune responses through the deletion or inactivation of conventional CD4 T cells and the formation or expansion of regulatory CD4 T cells (Treg). Although these "Ag-specific immunotherapy" (ASI) approaches are currently under clinical development to treat autoinflammatory conditions, efficacy and safety may be variable and unpredictable because of the diverse activation states of immune cells in subjects with autoimmune and allergic diseases. To reliably induce Ag-specific tolerance in patients, novel methods to control T cell responses during ASI are needed, and strategies that permanently increase Treg frequencies among Ag-specific CD4 T cells may provide long-lasting immunosuppression between treatments. In this study, we present an approach to durably increase the frequency of Ag-specific Treg in mice by administering ASI when Treg numbers are transiently increased with individual doses of a half-life-extended Treg-selective IL-2 mutein. Repeated weekly cycles of IL-2 mutein doses (day 0) followed by ASI (day 3) resulted in a 3- to 5-fold enrichment in Treg among Ag-responsive CD4 T cells. Expanded Ag-specific Treg persisted for more than 3 wk following treatment cessation, as well as through an inflammatory T cell response to an Ag-expressing virus. Combining Treg enrichment with ASI has the potential to durably treat autoimmune disease or allergy by increasing the Treg/conventional CD4 T cell ratio among autoantigen- or allergen-specific T cells.

Functional deficit in hippocampal activity during fear extinction recall in the single prolonged-stress model of PTSD in male rats.

To interrogate whether altered function of the hippocampal-mPFC circuit underlies the deficit in fear extinction recall in rats subjected to single-prolonged stress (SPS), changes in brain region-specific metabolic rate were measured in male rats (control and SPS treated). Brain region metabolic rates were quantified using uptake of 14C-2-deoxyglucose (14C-2DG) during fear memory formation, fear memory extinction and extinction recall. Control and SPS rats had similar regional brain activities at baseline. During extinction recall, 14C-2DG uptake decreased in hippocampal regions in control rats, but not in SPS rats. SPS rats also exhibited a significant deficiency in fear extinction recall, replicating a previously reported finding. Reduced hippocampal activity during fear extinction recall in control animals may reflect reduction in fear overgeneralization, thereby enabling discrimination between distinct contexts. In contrast, persistent levels of hippocampal activity in SPS-exposed male animals during fear extinction recall may reflect the dysfunctional persistence of fear overgeneralization. Future studies in females can test gender-specificity of these effects, with appropriate attention to luteal dependent effects on extinction of fear learning. Detailed knowledge of regional brain activities underlying stress-induced deficits in extinction recall may help identify therapeutic targets in PTSD.

Dopamine D1 Receptor Agonist PET Tracer Development: Assessment in Nonhuman Primates.

Non-catechol-based high-affinity selective dopamine D1 receptor (D1R) agonists were recently described, and candidate PET ligands were selected on the basis of favorable properties. The objective of this study was to characterize in vivo in nonhuman primates 2 novel D1R agonist PET radiotracers, racemic 18F-MNI-800 and its more active atropisomeric (-)-enantiomer, 18F-MNI-968. Methods: Ten brain PET experiments were conducted with 18F-MNI-800 on 2 adult rhesus macaques and 2 adult cynomolgus macaques, and 8 brain PET experiments were conducted with 18F-MNI-968 on 2 adult rhesus macaques and 2 adult cynomolgus macaques. PET data were analyzed with both plasma-input-based methods and reference-region-based methods. Whole-body PET images were acquired with 18F-MNI-800 from 2 adult rhesus macaques for radiation dosimetry estimates. Results:18F-MNI-800 and 18F-MNI-968 exhibited regional uptake consistent with D1R distribution. Specificity and selectivity were demonstrated by dose-dependent blocking with the D1 antagonist SCH-23390. 18F-MNI-968 showed a 30% higher specific signal than 18F-MNI-800, with a nondisplaceable binding potential of approximately 0.3 in the cortex and approximately 1.1 in the striatum. Dosimetry radiation exposure was favorable, with an effective dose of about 0.023 mSv/MBq. Conclusion:18F-MNI-968 has significant potential as a D1R agonist PET radiotracer, and further characterization in human subjects is warranted.

SPECT Imaging of Muscle Injury with [99mTc]MDP in a Mouse Model of Muscular Dystrophy.

PURPOSE: Tc-99m methylene diphosphonate ([99mTc]MDP) is an in vivo bone imaging agent that also accumulates in injured skeletal muscle cells. The objective of this study was to investigate if [99mTc]MDP could be used to detect muscle injury in the mdx mouse model of Duchenne muscular dystrophy (DMD). PROCEDURES: Static whole-body single-photon emission computed tomography/computed tomography (CT) scans were acquired at 2 h post-injection of [99mTc]MDP in two cohorts of animals at different sites: one cohort of mice at 6, 15, and 19 weeks of age, and a separate cohort at 16 weeks. The second cohort was also imaged with high-resolution CT at 8 weeks. RESULTS: mdx mice had higher [99mTc]MDP uptake and significantly higher [99mTc]MDP concentrations in muscle than controls. CONCLUSIONS: Higher uptake of [99mTc]MDP in muscle of mdx mice agrees with histological reports of muscle calcification in mdx mice, and suggests the potential translational use of [99mTc]MDP imaging for tracking DMD progression and therapeutic response.

Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

B cell adaptor for PI3-kinase (BCAP) modulates CD8+ effector and memory T cell differentiation.

CD8+ T cells respond to signals via the T cell receptor (TCR), costimulatory molecules, and immunoregulatory cytokines by developing into diverse populations of effector and memory cells. The relative strength of phosphoinositide 3-kinase (PI3K) signaling early in the T cell response can dramatically influence downstream effector and memory T cell differentiation. We show that initial PI3K signaling during T cell activation results in up-regulation of the signaling scaffold B cell adaptor for PI3K (BCAP), which further potentiates PI3K signaling and promotes the accumulation of CD8+ T cells with a terminally differentiated effector phenotype. Accordingly, BCAP-deficient CD8+ T cells have attenuated clonal expansion and altered effector and memory T cell development following infection with Listeria monocytogenes Thus, induction of BCAP serves as a positive feedback circuit to enhance PI3K signaling in activated CD8+ T cells, thereby acting as a molecular checkpoint regulating effector and memory T cell development.