Adapter CAR T cells to counteract T-cell exhaustion and enable flexible targeting in AML.

Although the landscape for treating acute myeloid leukemia (AML) patients has changed substantially in recent years, the majority of patients will eventually relapse and succumb to their disease. Allogeneic stem cell transplantation provides the best anti-AML treatment strategy, but is only suitable in a minority of patients. In contrast to B-cell neoplasias, chimeric antigen receptor (CAR) T-cell therapy in AML has encountered challenges in target antigen heterogeneity, safety, and T-cell dysfunction. We established a Fab-based adapter CAR (AdCAR) T-cell platform with flexibility of targeting and control of AdCAR T-cell activation. Utilizing AML cell lines and a long-term culture assay for primary AML cells, we were able to demonstrate AML-specific cytotoxicity using anti-CD33, anti-CD123, and anti-CLL1 adapter molecules in vitro and in vivo. Notably, we show for the first time the feasibility of sequential application of adapter molecules of different specificity in primary AML co-cultures. Importantly, using the AML platform, we were able to demonstrate that chronic T-cell stimulation and exhaustion can be counteracted through introduction of treatment-free intervals. As T-cell exhaustion and target antigen heterogeneity are well-known causes of resistance, the AdCAR platform might offer effective strategies to ameliorate these limitations.

A fast activating presynaptic reuptake current during serotonergic transmission in identified neurons of Hirudo.

An electrogenic serotonin (5-HT) uptake process was characterized in the serotonergic Retzius-P cell synapse of the leech, and the simultaneous activation of this presynaptic reuptake and the postsynaptic response was monitored during evoked transmitter release. A presynaptic, Na(+)-dependent inward current upon application of 5-HT was isolated at membrane potentials between -80 and +60 mV. Its identification as a transmitter uptake current was confirmed by monitoring accumulation of the autofluorescent 5-HT analog 5,7-dihydroxytryptamine during activation of this current. To study the kinetics of 5-HT reuptake in functional synapses, transmitter release was stimulated by flash photolysis of the Ca(2+)-caging DM-nitrophen. The results demonstrate that reuptake activates with a minimal delay of less than a millisecond during synaptic transmission. It acts as a rapid transmitter removal system to determine the time course of the postsynaptic response and monitors the kinetics of transmitter clearance at the synaptic site.

Emergence of input specificity of ltp during development of retinotectal connections in vivo.

Input specificity of activity-induced synaptic modification was examined in the developing Xenopus retinotectal connections. Early in development, long-term potentiation (LTP) induced by theta burst stimulation (TBS) at one retinal input spreads to other unstimulated converging inputs on the same tectal neuron. As the animal develops, LTP induced by the same TBS becomes input specific, a change that correlates with the increased complexity of tectal dendrites and more restricted distribution of dendritic Ca(2+) evoked by each retinal input. In contrast, LTP induced by 1 Hz correlated pre- and postsynaptic spiking is input specific throughout the same developmental period. Thus, input specificity of LTP emerges with neural development and depends on the pattern of synaptic activity.

A low-cost UV laser for flash photolysis of caged compounds.

Photolysis of caged compounds has become a standard tool for the rapid application of bioactive molecules. In principle this technique also allows to apply substances in a spatially very restricted manner. An important practical limitation for such experiments, however, is the high cost of UV lasers. Here we describe the assembly of an inexpensive pulsed nitrogen laser which is suitable for photolysis experiments. The laser which can be constructed in less than 1 week and for less than US$ 500 emits light pulses with a duration of approximately 5 ns, an energy of up to 200 microJ (= 40 kW) and a wavelength of 337 nm. Its beam can be focused to roughly 30 microns, a firing frequency of up to 50 Hz can be achieved, and electrical artifacts are minimal. These specifications make the laser optimally suited for most photolysis experiments. Its low price and ease of use should make the technique of spatially restricted flash photolysis amenable to many laboratories.

Different effects of baclofen and GTP gamma S on voltage-activated Ca2+ currents in rat hippocampal neurons in vitro.

Reduction of voltage-activated Ca2+ currents by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) through ultraviolet (UV) photolysis of the caged compound, is followed by a re-augmentation to control levels within 10 min, independently of the divalent cation used. The Ca2+ current inhibition by the gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen, which is also thought to be mediated by a GTP-binding protein (G-protein), is potentiated when GTP gamma S is uncaged during agonist superfusion. The authors suggest that GTP gamma S activates G-protein-dependent pathways that are not activated by the baclofen receptor.

Synapse specificity of long-term potentiation breaks down at short distances.

Long-term potentiation (LTP), the long-lasting increase in synaptic transmission, has been proposed to be a cellular mechanism essential for learning and memory, neuronal development, and circuit reorganization. In the original theoretical and experimental work it was assumed that only synapses that had experienced concurrent pre- and postsynaptic activity are subject to synaptic modification. It has since been shown, however, that LTP is also expressed in synapses on neighbouring neurons that have not undergone the induction procedure. Yet, it is still believed that this spread of LTP is limited to adjacent postsynaptic cells, and does not occur for synapses on neighbouring input fibres. However, for technical reasons, tests for 'input specificity' were always done for synapses relatively far apart. Here we have used a new local superfusion technique, which allowed us to assess the synaptic specificity of LTP with a spatial resolution of approximately 30 microm. Our results indicate that there is no input specificity at a distance of less than 70 microm. Synapses in close proximity to a site of potentiation are also potentiated regardless of their own history of activation, whereas synapses far away show no potentiation.

Dendritic spine changes associated with hippocampal long-term synaptic plasticity.

Long-term enhancement of synaptic efficacy in the hippocampus is an important model for studying the cellular mechanisms of neuronal plasticity, circuit reorganization, and even learning and memory. Although these long-lasting functional changes are easy to induce, it has been very difficult to demonstrate that they are accompanied or even caused by morphological changes on the subcellular level. Here we combined a local superfusion technique with two-photon imaging, which allowed us to scrutinize specific regions of the postsynaptic dendrite where we knew that the synaptic changes had to occur. We show that after induction of long-lasting (but not short-lasting) functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite, whereas in control regions on the same dendrite or in slices where long-term potentiation was blocked, no significant spine growth occurred.

Synaptic current kinetics in a solely AMPA-receptor-operated glutamatergic synapse formed by rat retinal ganglion neurons.

1. Postnatal rat retinal ganglion cells (RGCs) can be maintained and identified in dissociated long-term culture. After 4-7 days in vitro they form glutamatergic synapses with other RGCs or putative amacrine cells. Here we intended to characterize the postsynaptic features of these in vitro synapses. 2. Pair patch-clamp recordings in the whole cell configuration were performed to study the properties of synaptic glutamate receptors. Immunohistochemically and physiologically identified RGCs were activated by short depolarizing voltage steps. This elicited glutamatergic excitatory postsynaptic currents (EPSCs) in coupled neurons. At room temperature, evoked EPSCs (eEPSCs) had latencies between 3 and 7 ms and amplitudes between 36.4 and 792.6 pA. 3. Postsynaptic neurons were electrotonically compact and therefore well suited for analysis of fast synaptic events. All cells were responsive to exogenous glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA). The current-voltage relationships of AMPA-activated currents were linear, whereas NMDA-induced whole cell currents displayed the typical characteristics including a negative slope conductance in the presence of Mg2+. In contrast to AMPA-activated currents, NMDA-activated currents had the usual slow onset and decay. 4. RGCs obviously failed to generate NMDA-receptor-mediated EPSCs, because all postsynaptic cells lacked a slow current component even in the absence of added Mg2+ and in the presence of glycine. Retinal eEPSCs were completely blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX). 5. eEPSCs as well as spontaneous EPSCs (sEPSCs) were characterized by a very rapid time course. In eEPSCs, 20-80% rise times and time constants of decay (tau DS) were on average 0.64 and 1.96 ms, respectively. eEPSCs were extremely fast, with average rise times of 0.34 ms and tau DS of 1.20 ms. The latter numbers closely correspond to the values obtained for DNQX-sensitive miniature EPSC (mEPSC) in postnatal day 5 rat RGCs in situ. 6. To clarify whether the decay of fast AMPA-receptor-mediated EPSCs of retinal neurons was determined by the onset of glutamate receptor desensitization, we compared the decay of sEPSCs with the decay of the glutamate response of excised out-side-out membrane patches. Glutamate-activated currents were elicited by a rapid superfusion device (time constant of rise = 0.7 ms). The response to 1 mM of glutamate decayed 2 to 4 times more slowly than the sEPSCs. 7. These results suggest that desensitization did not limit the rate of decay of purely AMPA-mediated EPSCs in response to ganglion cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Fast local superfusion technique.

A system for rapid, local superfusion of cultured neurones and their neurites with various different test drugs is elucidated. An area of down to 30 micron diameter was superfused with the aid of two micropipettes, one for delivering the test solution and the other for its removal. Active removal of solution within the deadspace of the delivery pipette guarantees, on the one hand, fast and flexible pressure control and, on the other, enables the quick exchange (<1 s) of multiple solutions. By increasing the pressure in the superfusion pipette, the laminar stream between the pipettes was forced down onto the cell layer. The change from bath to superfusion solutions, evaluated by liquid junction potential changes, occurs in the order of 1 ms.