ABSTRACT
Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.
Subject(s)
Immunological Synapses/immunology , Neoplasms/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , CD3 Complex , Cell Adhesion , Cell Death , Cell Line, Tumor , Computational Biology , Cytokines/metabolism , Dyneins/chemistry , Ligands , Lymphocyte Activation , Lymphocyte Function-Associated Antigen-1/immunology , Mice , Microtubules/metabolism , Signal TransductionABSTRACT
Time-lapse video microscopy allows analysis of the interaction between individual CTLs and adherent peptide-pulsed targets, from contact, to lymphocyte detachment, APC rounding, phosphatidylserine exposure and finally loss of plasma membrane integrity characteristic of end-stage apoptosis. Using in vitro-stimulated effectors specific for the ovalbumin K(b)OVA(257) (OT-I) and influenza A virus D(b)NP(366) and D(b)PA(224) epitopes, no significant correlation was found between the duration of CTL contact and the time to phosphatidylserine exposure or loss of membrane integrity. Furthermore, there were minimal indications that transgenic T cells specific for the K(b)OVA(257) epitope (TCR) diversity had any effect. However, when the analysis was repeated with D(b)NP(366) and D(b)PA(224)-specific CTLs recovered directly from the lungs of mice with influenza pneumonia, the lower avidity D(b)NP(366)-specific set was found to elute much more quickly. Shorter contact time may allow individual CTLs to lyse more targets, suggesting that lower TCR/epitope avidity may be more beneficial than higher epitope avidity for cell-mediated immunity.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , Microscopy, Video/methods , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , CD8-Positive T-Lymphocytes/physiology , Cells, Cultured , Influenza A virus/immunology , Mice , Ovalbumin/immunology , T-Lymphocytes, Cytotoxic/physiologyABSTRACT
This study was conducted to determine whether alcoholics' memory difficulties are due, in part, to access (retrieval) or to availability (retention) deficits. Forty-four alcoholics (n = 20 females) and 44 controls (n = 22 females) learned a paired associate list consisting of 12 adjective-CCC trigram pairs. Half of the subjects in each group learned the list to a low degree of learning (DOL; 4/12 pairs); the remainder to a high DOL (8/12 pairs). Two distinct environmental contexts (providing implicit cues) were used during acquisition. Subjects then completed a cued recall (an explicit cue) test in either the same or a different room. Alcoholics were significantly inferior in the acquisition phase on trials required to reach criterion, regardless of DOL required [F(1,68) = 10.92, p = 0.002]. The main effect for implicit cuing was not significant; similarly, there were no significant interactions. In contrast, the explicit cue manipulation produced a significant group x DOL interaction on the number of trigrams correctly recalled [F = (1,77) = 6.38, p = 0.01]; alcoholics' recall did not benefit from the higher DOL in contrast to a significant improvement in recall by controls. The failure of alcoholics to demonstrate improvement with higher levels of learning is consistent with a deficit in the availability of information. The results confirm previous reports of recovering alcoholics' verbal learning and memory dysfunction, and suggest that these deficits may be attributed, in part, to a deficit in the availability of information (retention).
Subject(s)
Alcohol Amnestic Disorder/diagnosis , Cues , Mental Recall , Paired-Associate Learning , Retention, Psychology , Adult , Alcohol Amnestic Disorder/psychology , Alcoholism/psychology , Alcoholism/rehabilitation , Female , Humans , Male , Middle AgedABSTRACT
A novel series of 5-(1,1-dioxo-1,2,5-thiadiazolidin-2-yl)tryptamines was designed, synthesized, and evaluated as 5-HT1D receptor agonists. Compounds such as 8d,f,k were identified which had comparable affinity, potency, and receptor selectivity to that of the antimigraine drug sumatriptan. Both 8d,k were found to be well absorbed in the rat with oral bioavailabilities of 66% and 62%, respectively. Additionally, 8d was found to be selective over other non-serotonergic receptors and exhibited relatively low central nervous system penetration.
