CD39 Is Expressed on Functional Effector and Tissue-resident Memory CD8+ T Cells.

The ecto-ATPase CD39 is expressed on exhausted CD8+ T cells in chronic viral infection and has been proposed as a marker of tumor-specific CD8+ T cells in cancer, but the role of CD39 in an effector and memory T cell response has not been clearly defined. We report that CD39 is expressed on Ag-specific CD8+ short-lived effector cells, while it's co-ectoenzyme, CD73, is found on memory precursor effector cells (MPECs) in vivo. Inhibition of CD39 enzymatic activity during in vitro T cell priming enhances MPEC differentiation in vivo after transfer and infection. The enriched MPEC phenotype is associated with enhanced tissue resident memory T cell (TRM cell) establishment in the brain and salivary gland following an acute intranasal viral infection, suggesting that CD39 ATPase activity plays a role in memory CD8+ T cell differentiation. We also show that CD39 is expressed on human and murine TRM cells across several nonlymphoid tissues and melanoma, whereas CD73 is expressed on both circulating and resident memory subsets in mice. In contrast to exhausted CD39+ T cells in chronic infection, CD39+ TRM cells are fully functional when stimulated ex vivo with cognate Ag, further expanding the identity of CD39 beyond a T cell exhaustion marker.

Functional virus-specific memory T cells survey glioblastoma.

Glioblastoma multiforme (GBM) is among the most aggressive, treatment-resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host and are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8 + T cells expressing tissue-resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific memory T cells through intratumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to antineoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific memory T cells are a significant part of the glioblastoma immune microenvironment and may be leveraged to promote anti-tumoral immunity.

Persistent Interneuronopathy in the Prefrontal Cortex of Young Adult Offspring Exposed to Ethanol In Utero.

Gestational exposure to ethanol has been reported to alter the disposition of tangentially migrating GABAergic cortical interneurons, but much remains to be elucidated. Here we first established the migration of interneurons as a proximal target of ethanol by limiting ethanol exposure in utero to the gestational window when tangential migration is at its height. We then asked whether the aberrant tangential migration of GABAergic interneurons persisted as an enduring interneuronopathy in the medial prefrontal cortex (mPFC) later in the life of offspring prenatally exposed to ethanol. Time pregnant mice with Nkx2.1Cre/Ai14 embryos harboring tdTomato-fluorescent medial ganglionic eminence (MGE)-derived cortical GABAergic interneurons were subjected to a 3 day binge-type 5% w/w ethanol consumption regimen from embryonic day (E) 13.5-16.5, spanning the peak of corticopetal interneuron migration in the fetal brain. Our binge-type regimen increased the density of MGE-derived interneurons in the E16.5 mPFC. In young adult offspring exposed to ethanol in utero, this effect persisted as an increase in the number of mPFC layer V parvalbumin-immunopositive interneurons. Commensurately, patch-clamp recording in mPFC layer V pyramidal neurons uncovered enhanced GABA-mediated spontaneous and evoked synaptic transmission, shifting the inhibitory/excitatory balance toward favoring inhibition. Furthermore, young adult offspring exposed to the 3 day binge-type ethanol regimen exhibited impaired reversal learning in a modified Barnes maze, indicative of decreased PFC-dependent behavioral flexibility, and heightened locomotor activity in an open field arena. Our findings underscore that aberrant neuronal migration, inhibitory/excitatory imbalance, and thus interneuronopathy contribute to indelible abnormal cortical circuit form and function in fetal alcohol spectrum disorders. SIGNIFICANCE STATEMENT: The significance of this study is twofold. First, we demonstrate that a time-delimited binge-type ethanol exposure in utero during early gestation alters corticopetal tangential migration of GABAergic interneurons in the fetal brain. Second, our study is the first to integrate neuroanatomical, electrophysiological, and behavioral evidence that this "interneuronopathy" persists in the young adult offspring and contributes to enduring changes in (1) the distribution of parvalbumin-expressing GABAergic cortical interneurons in the medial prefrontal cortex, (2) GABA-mediated synaptic transmission that resulted in an inhibitory/excitatory synaptic imbalance, and (3) behavioral flexibility. These findings alert women of child-bearing age that fetal alcohol spectrum disorders can be rooted very early in fetal brain development, and reinforce evidence-based counseling against binge drinking even at the earliest stages of pregnancy.

Chronic Gestational Exposure to Ethanol Leads to Enduring Aberrances in Cortical Form and Function in the Medial Prefrontal Cortex.

BACKGROUND: Exposure to ethanol (EtOH) in utero alters the disposition of tangentially migrating GABAergic interneurons in the fetal brain. The medial ganglionic eminence (MGE) gives rise to a large portion of cortical GABAergic interneurons, including the parvalbumin-expressing interneurons that shape and contribute to inhibitory/excitatory (I/E) balance of the intracortical circuit. Here, we investigated in the mouse medial prefrontal cortex (mPFC) the hypothesis that low levels of maternal EtOH consumption from closure of the neural tube embryonic day (E) 9.5 until birth result in an enduring interneuronopathy. METHODS: Pregnant mice were subjected to a 2% w/w EtOH consumption regimen starting at neural tube closure and ending at parturition. Neurogenesis in the MGE was assessed by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence at E12.5. The count and distribution of parvalbumin-expressing interneurons were determined in adult animals, and patch clamp electrophysiology was performed to determine GABAergic function and I/E balance. Open-field behavior in adult mice was assessed to determine whether the EtOH-exposed cohort displayed a lasting alteration in exploratory behavior. RESULTS: In embryos exposed to EtOH in utero, we found increased BrdU labeling in the MGE, pointing to increased neurogenesis. Adult mice prenatally exposed to EtOH were hyperactive, and this was associated with an increase in parvalbumin-expressing GABAergic interneurons in the mPFC. In addition, prenatal EtOH exposure altered the balance between spontaneous inhibitory and excitatory synaptic input and attenuated GABAergic tone in layer V mPFC pyramidal neurons in juvenile mice. CONCLUSIONS: These findings underscore that altered migration of GABAergic interneurons contributes to the EtOH-induced aberration of cortical development and that these effects persist into adulthood as altered cortical form and function.

Effects of ethanol exposure in utero on Cajal-Retzius cells in the developing cortex.

BACKGROUND: Prenatal exposure to ethanol exerts teratogenic effects on the developing brain. Here, we tested the hypothesis that exposure to ethanol in utero alters the disposition of Cajal-Retzius cells that play a key role in orchestrating proliferation, migration, and laminar integration of cortical neurons in the embryonic cortex. METHODS: Pregnant Ebf2-EGFP mice, harboring EGFP-fluorescent Cajal-Retzius cells, were subjected to a 2% w/w ethanol consumption regimen starting at neural tube closure and lasting throughout gestation. Genesis of Cajal-Retzius cells was assessed by means of 5-bromo-2-deoxyuridine (BrdU) immunofluorescence at embryonic day 12.5, their counts and distribution were determined between postnatal day (P)0 and P4, patch clamp electrophysiology was performed between P2 and P3 to analyze GABA-mediated synaptic activity, and open-field behavioral testing was conducted in P45-P50 adolescents. RESULTS: In Ebf2-EGFP embryos exposed to ethanol in utero, we found increased BrdU labeling and expanded distribution of Cajal-Retzius cells in the cortical hem, pointing to increased genesis and proliferation. Postnatally, we found an increase in Cajal-Retzius cell number in cortical layer I. In addition, they displayed altered patterning of spontaneous GABA-mediated synaptic barrages and enhanced GABA-mediated synaptic activity, suggesting enhanced GABAergic tone. CONCLUSIONS: These findings, together, underscore that Cajal-Retzius cells contribute to the ethanol-induced aberration of cortical development and abnormal GABAergic neurotransmission at the impactful time when intracortical circuits form.

Alarm functions of PD-1+ brain resident memory T cells.

Resident memory T cells (T RM ) have been described in barrier tissues as having a 'sensing and alarm' function where, upon sensing cognate antigen, they alarm the surrounding tissue and orchestrate local recruitment and activation of immune cells. In the immunologically unique and tightly restricted CNS, it remains unclear if and how brain T RM , which express the inhibitory receptor PD-1, alarm the surrounding tissue during antigen re-encounter. Here, we reveal that T RM are sufficient to drive the rapid remodeling of the brain immune landscape through activation of microglia, DCs, NK cells, and B cells, expansion of Tregs, and recruitment of macrophages and monocytic dendritic cells. Moreover, we report that while PD-1 restrains granzyme B expression by reactivated brain T RM , it has no effect on cytotoxicity or downstream alarm responses. We conclude that T RM are sufficient to trigger rapid immune activation and recruitment in the CNS and may have an unappreciated role in driving neuroinflammation.

CD39 is expressed on functional effector and tissue resident memory CD8+ T cells.

The ecto-ATPase CD39 is expressed on exhausted CD8+ T cells in chronic viral infection and has been proposed as a marker of tumor-specific CD8+ T cells in cancer, but the role of CD39 in an effector and memory T cell response has not been clearly defined. We report that CD39 is expressed on antigen-specific CD8+ short-lived effector cells (SLECs), while it's co-ecto-enzyme, CD73, is found on memory precursor effector cells (MPEC) in vivo . Inhibition of CD39 enzymatic activity during in vitro T cell priming enhances MPEC differentiation in vivo after transfer and infection. The enriched MPEC phenotype is associated with enhanced tissue resident memory (T RM ) establishment in the brain and salivary gland following an acute intranasal viral infection, suggesting that CD39 ATPase activity plays a role in memory CD8+ T cell differentiation. We also show that CD39 is expressed on human and murine T RM across several non-lymphoid tissues and melanoma, while CD73 is expressed on both circulating and resident memory subsets in mice. In contrast to exhausted CD39+ T cells in chronic infection, CD39+ T RM are fully functional when stimulated ex vivo with cognate antigen. This work further expands the identity of CD39 beyond a T cell exhaustion marker.

Targeting the somatosensory system with AAV9 and AAV2retro viral vectors.

Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products.

AAV-Mediated Gene Delivery to the Spinal Cord by Intrathecal Injection.

Gene therapy targeting the spinal cord is an important tool for analyzing mechanisms of nervous system diseases and the development of gene therapies. Analogous to a lumbar puncture in humans, the rodent spinal cord can be accessed through an efficient, noninvasive injection. Here we describe a method for AAV-mediated gene transfer to cells of the spinal cord by intrathecal injection of small quantities of AAV vector.

Involvement of the VGF-derived peptide TLQP-62 in nerve injury-induced hypersensitivity and spinal neuroplasticity.

Neuroplasticity in the dorsal horn after peripheral nerve damage contributes critically to the establishment of chronic pain. The neurosecretory protein VGF (nonacronymic) is rapidly and robustly upregulated after nerve injury, and therefore, peptides generated from it are positioned to serve as signals for peripheral damage. The goal of this project was to understand the spinal modulatory effects of the C-terminal VGF-derived peptide TLQP-62 at the cellular level and gain insight into the function of the peptide in the development of neuropathic pain. In a rodent model of neuropathic pain, we demonstrate that endogenous levels of TLQP-62 increased in the spinal cord, and its immunoneutralization led to prolonged attenuation of the development of nerve injury-induced hypersensitivity. Using multiphoton imaging of submaximal glutamate-induced Ca responses in spinal cord slices, we demonstrate the ability of TLQP-62 to potentiate glutamatergic responses in the dorsal horn. We further demonstrate that the peptide selectively potentiates responses of high-threshold spinal neurons to mechanical stimuli in singe-unit in vivo recordings. These findings are consistent with a function of TLQP-62 in spinal plasticity that may contribute to central sensitization after nerve damage.