CD4+ CAR T-cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR T-cell therapy.

ABSTRACT: Multiple myeloma is characterized by frequent clinical relapses after conventional therapy. Recently, chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, although >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune-intrinsic mechanisms may contribute to this resistance. Although there were no preexisting T-cell features associated with clinical outcomes, we found that patients with a durable response to CAR T-cell treatment had greater persistence of their CAR T cells than patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T-effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR T cells. These cells expand in vivo early after infusion but express exhaustion markers (hepatitis A virus cellular receptor 2 [HAVCR2] and T-cell immunoglobulin and mucin domain-containing-3 [TIGIT]) and remain polyclonal. Finally, we demonstrate that nonclassical monocytes are enriched in the myeloma niche and may induce CAR T-cell dysfunction through mechanisms that include transforming growth factor ß. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR T-cell therapy.

NAIR: Network Analysis of Immune Repertoire.

T cells represent a crucial component of the adaptive immune system and mediate anti-tumoral immunity as well as protection against infections, including respiratory viruses such as SARS-CoV-2. Next-generation sequencing of the T-cell receptors (TCRs) can be used to profile the T-cell repertoire. We developed a customized pipeline for Network Analysis of Immune Repertoire (NAIR) with advanced statistical methods to characterize and investigate changes in the landscape of TCR sequences. We first performed network analysis on the TCR sequence data based on sequence similarity. We then quantified the repertoire network by network properties and correlated it with clinical outcomes of interest. In addition, we identified (1) disease-specific/associated clusters and (2) shared clusters across samples based on our customized search algorithms and assessed their relationship with clinical outcomes such as recovery from COVID-19 infection. Furthermore, to identify disease-specific TCRs, we introduced a new metric that incorporates the clonal generation probability and the clonal abundance by using the Bayes factor to filter out the false positives. TCR-seq data from COVID-19 subjects and healthy donors were used to illustrate that the proposed approach to analyzing the network architecture of the immune repertoire can reveal potential disease-specific TCRs responsible for the immune response to infection.

A Predictive Model of Capillary Forces and Contact Diameters between Two Plates Based on Artificial Neural Network.

Many efforts have been devoted to the forecasting of the capillary force generated by capillary adsorption between solids, which is fundamental and essential in the fields of micro-object manipulation and particle wetting. In this paper, an artificial neural network (ANN) model optimized by a genetic algorithm (GA-ANN) was proposed to predict the capillary force and contact diameter of the liquid bridge between two plates. The mean square error (MSE) and correlation coefficient (R2) were employed to evaluate the prediction accuracy of the GA-ANN model, theoretical solution method of the Young-Laplace equation and simulation approach based on the minimum energy method. The results showed that the values of MSE of capillary force and contact diameter using GA-ANN were 10.3 and 0.0001, respectively. The values of R2 were 0.9989 and 0.9977 for capillary force and contact diameter in regression analysis, respectively, demonstrating the accuracy of the proposed predictive model. The sensitivity analysis was conducted to investigate the influence of input parameters, including liquid volume and separation distance, on the capillary force and contact diameter. The liquid volume and separation distance played dominant roles in affecting the capillary force and contact diameter.

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce T cell paralysis.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n = 9) and matched healthy donors (n = 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ T cells and induce SOCS3 expression in CD4+ T cells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce T cell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.

Flexible Magnetic Micropartners for Micromanipulation at Interfaces.

Microrobots working at liquid surfaces have immense potential for micromanipulation in tight and enclosed spaces, whereas constructing agile and functional microrobots with simple structures at liquid surfaces is a great challenge. Herein, a pair of magnetic circular microdisks working as partners at ethylene glycol (EG) surfaces are proposed in order to accomplish flexible locomotion and in situ micromanipulation tasks. The microdisks can be controlled to connect and separate by modulating the orientation of the applied magnetic field. After the two disks connect as an entity, they are transformed into micropartners under an oscillating magnetic field in 3D space. By changing the vertical component of the oscillating field, the micropartners can obtain controllable propulsion through paddling and wriggling modes, and the locomotion speed can reach approximately two body lengths per second. They can also climb a meniscus, and even crawl on a solid surface in a liquid. Finally, this pair of micropartners is demonstrated as a flexible microgripper to implement manipulations at the liquid surfaces, including cargo capture, delivery along prescribed paths, and release.

Novel Ensemble Feature Selection Approach and Application in Repertoire Sequencing Data.

The T and B cell repertoire make up the adaptive immune system and is mainly generated through somatic V(D)J gene recombination. Thus, the VJ gene usage may be a potential prognostic or predictive biomarker. However, analysis of the adaptive immune system is challenging due to the heterogeneity of the clonotypes that make up the repertoire. To address the heterogeneity of the T and B cell repertoire, we proposed a novel ensemble feature selection approach and customized statistical learning algorithm focusing on the VJ gene usage. We applied the proposed approach to T cell receptor sequences from recovered COVID-19 patients and healthy donors, as well as a group of lung cancer patients who received immunotherapy. Our approach identified distinct VJ genes used in the COVID-19 recovered patients comparing to the healthy donors and the VJ genes associated with the clinical response in the lung cancer patients. Simulation studies show that the ensemble feature selection approach outperformed other state-of-the-art feature selection methods based on both efficiency and accuracy. It consistently yielded higher stability and sensitivity with lower false discovery rates. When integrated with different classification methods, the ensemble feature selection approach had the best prediction accuracy. In conclusion, the proposed novel approach and the integration procedure is an effective feature selection technique to aid in correctly classifying different subtypes to better understand the signatures in the adaptive immune response associated with disease or the treatment in order to improve treatment strategies.

Capillary Forces between Concave Gripper and Spherical Particle for Micro-Objects Gripping.

The capillary action between two solid surfaces has drawn significant attention in micro-objects manipulation. The axisymmetric capillary bridges and capillary forces between a spherical concave gripper and a spherical particle are investigated in the present study. A numerical procedure based on a shooting method, which consists of double iterative loops, was employed to obtain the capillary bridge profile and bring the capillary force subject to a constant volume condition. Capillary bridge rupture was characterized using the parameters of the neck radius, pressure difference, half-filling angle, and capillary force. The effects of various parameters, such as the contact angle of the spherical concave gripper, the radius ratio, and the liquid bridge volume on the dimensionless capillary force, are discussed. The results show that the radius ratio has a significant influence on the dimensionless capillary force for the dimensionless liquid bridge volumes of 0.01, 0.05, and 0.1 when the radius ratio value is smaller than 10. The effectiveness of the theorical approach was verified using simulation model and experiments.

Mutations in Metabotropic Glutamate Receptor 1 Contribute to Natural Short Sleep Trait.

Sufficient and efficient sleep is crucial for our health. Natural short sleepers can sleep significantly shorter than the average population without a desire for more sleep and without any obvious negative health consequences. In searching for genetic variants underlying the short sleep trait, we found two different mutations in the same gene (metabotropic glutamate receptor 1) from two independent natural short sleep families. In vitro, both of the mutations exhibited loss of function in receptor-mediated signaling. In vivo, the mice carrying the individual mutations both demonstrated short sleep behavior. In brain slices, both of the mutations changed the electrical properties and increased excitatory synaptic transmission. These results highlight the important role of metabotropic glutamate receptor 1 in modulating sleep duration.

Mutant neuropeptide S receptor reduces sleep duration with preserved memory consolidation.

Sleep is a crucial physiological process for our survival and cognitive performance, yet the factors controlling human sleep regulation remain poorly understood. Here, we identified a missense mutation in a G protein-coupled neuropeptide S receptor 1 (NPSR1) that is associated with a natural short sleep phenotype in humans. Mice carrying the homologous mutation exhibited less sleep time despite increased sleep pressure. These animals were also resistant to contextual memory deficits associated with sleep deprivation. In vivo, the mutant receptors showed increased sensitivity to neuropeptide S exogenous activation. These results suggest that the NPS/NPSR1 pathway might play a critical role in regulating human sleep duration and in the link between sleep homeostasis and memory consolidation.

A class of circadian long non-coding RNAs mark enhancers modulating long-range circadian gene regulation.

Circadian rhythm exerts its influence on animal physiology and behavior by regulating gene expression at various levels. Here we systematically explored circadian long non-coding RNAs (lncRNAs) in mouse liver and examined their circadian regulation. We found that a significant proportion of circadian lncRNAs are expressed at enhancer regions, mostly bound by two key circadian transcription factors, BMAL1 and REV-ERBα. These circadian lncRNAs showed similar circadian phases with their nearby genes. The extent of their nuclear localization is higher than protein coding genes but less than enhancer RNAs. The association between enhancer and circadian lncRNAs is also observed in tissues other than liver. Comparative analysis between mouse and rat circadian liver transcriptomes showed that circadian transcription at lncRNA loci tends to be conserved despite of low sequence conservation of lncRNAs. One such circadian lncRNA termed lnc-Crot led us to identify a super-enhancer region interacting with a cluster of genes involved in circadian regulation of metabolism through long-range interactions. Further experiments showed that lnc-Crot locus has enhancer function independent of lnc-Crot's transcription. Our results suggest that the enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs.

Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression.

Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure.

Oscillating primary transcripts harbor miRNAs with circadian functions.

The roles of miRNAs as important post-transcriptional regulators in the circadian clock have been suggested in several studies. But the search for circadian miRNAs has led to disparate results. Here we demonstrated that at least 57 miRNA primary transcripts are rhythmically transcribed in mouse liver. Most of these transcripts are under the regulation of circadian transcription factors such as BMAL1/CLOCK and REV-ERBα/ß. However, the mature miRNAs derived from these transcripts are either not oscillating or oscillating at low amplitudes, which could explain the inconsistency of different circadian miRNA studies. In order to show that these circadian primary transcripts can give rise to miRNAs with circadian functions, we over-expressed one of them, miR-378, in mouse by adenovirus injection. We found a significant over-representation of circadian oscillating genes under-expressed by miR-378 over-expression in liver. In particular, we observed that miR-378 modulates the oscillation amplitudes of Cdkn1a in the control of cell cycle and Por in the regulation of oxidation reduction by forming partnership with different circadian transcription factors. Our study suggests that circadian transcription of miRNA at primary transcript level can be a good indicator for circadian miRNA functions.

A vacuum microgripping tool with integrated vibration releasing capability.

Pick-and-place of micro-objects is a basic task in various micromanipulation demands. Reliable releasing of micro-objects is usually disturbed due to strong scale effects. This paper focuses on a vacuum micro-gripper with vibration releasing functionality, which was designed and assembled for reliable micromanipulation tasks. Accordingly, a vibration releasing strategy of implementing a piezoelectric actuator on the vacuum microgripping tool is presented to address the releasing problem. The releasing mechanism was illustrated using a dynamic micro contact model. This model was developed via theoretical analysis, simulations and pull-off force measurement using atomic force microscopy. Micromanipulation experiments were conducted to verify the performance of the vacuum micro-gripper. The results show that, with the assistance of the vibration releasing, the vacuum microgripping tool can achieve reliable release of micro-objects. A releasing location accuracy of 4.5±0.5 µm and a successful releasing rate of around 100% (which is based on 110 trials) were achieved for manipulating polystyrene microspheres with radius of 35-100 µm.