Biology subjects

Kaur, A.

Publications and source records attributed to Kaur, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Aberrant NOTUM+ Program Induced in LGR5+ Crypt Base Columnar Cells Maintains an Immunosuppressive Niche in Colorectal Cancer

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with treatment failure largely driven by cancer stem-like cells that resist conventional chemoradiation and subsequently initiate tumor recurrence. While immune checkpoint blockade is effective in microsatellite instability-high (MSI-H) CRCs, the majority of CRCs are microsatellite stable (MSS) and exhibit immune exclusion, rendering them refractory to immunotherapy. Here, we identify a previously uncharacterized cancer cell subtype, which we term cancerous Crypt Base Columnar (canCBC) cells. These cells transcriptionally resemble normal LGR5+ CBC cells but activate an aberrant WNT/{beta}-catenin signalling inhibitory program, marked by NOTUM expression. We show that canCBC cells are specifically enriched in MSS tumors, where their presence correlates with reduced CD8 T cell infiltration, broader immune exclusion, and a propensity for regional lymphatic dissemination. Consistently, targeted ablation of canCBCs enhances the tumor-clearing potential of CD8 T cells. This study identifies a novel therapeutic target for overcoming immune exclusion and improving immunotherapy responses in MSS CRCs.

cancer biology↗

bioRxiv2025

A nonhuman primate model mirrors human congenital cytomegalovirus infection and reveals a spectrum of vertical transmission outcomes

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform design of a maternal vaccine remain elusive. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection during pregnancy in an immune competent nonhuman primate (NHP) model. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) CMV-naive rhesus macaque dams inoculated intravenously with RhCMV in early second trimester gestation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). Placental RhCMV transmission was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. CMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection as evidenced by RhCMV infection at the maternal-fetal interface in all 12 dams, along with significantly reduced placental efficiency and fetal growth metrics compared to gestation-matched control pregnancies. This NHP model recapitulates key aspects of human cCMV and provides new insight into barriers and biomarkers of successful vertical transmission. One sentence summary: The nonhuman primate model mirrors the epidemiology of human congenital CMV (cCMV) after primary infection and reveals its transmission bottlenecks.

immunology↗

bioRxiv2025

Optimizing Transfection Efficiency in Primary Human Splenic Fibroblast Cells

Transfection, a fundamental molecular biology technique, is crucial in introducing foreign DNA into cells. Despite its pivotal importance, mastering this technique remains a formidable challenge. In this study, we employed the calcium phosphate reagent and conducted a systematic exploration through time-dependent experiments to assess its impact on human primary splenic fibroblast cells (HSFC). The results precisely delineate optimal time frames for achieving enhanced transfection efficiency, offering conclusive evidence for the efficacy of calcium phosphate in transient transfection within the cells. The data significantly advances our comprehension of HSFC reactions, contributing to refining transfection methodologies with broader implications for biomedical applications.

cell biology↗

bioRxiv2025

Detection of resistance in Phytophthora infestans to the carboxylic acid amide (CAA) fungicides using digital droplet PCR.

BackgroundPotato late blight caused by Phytophthora infestans remains the greatest biotic threat to potato production globally. In northern Europe to prevent the disease and associated yield losses fungicides are heavily relied upon, with multiple applications required during the potato growing season. Unfortunately, such intensive fungicide usage has the potential to compromise control efficacy as it puts in place immense selective pressure for the emergence and spread of fungicide resistant strains of P. infestans. In recent years this scenario has been realised, with the emergence of strains resistant to the carboxylic acid amide (CAA) fungicides. As resistance to the CAA fungicides in P. infestans has been confirmed to result from changes in the pathogens cellulose synthase A3 gene (CesA3), specifically at amino acid position 1105, it opens the possibility to develop molecular tools to rapidly monitor populations of the pathogen for the resistance allele. ResultsUsing the Stilla nacia 3-colour system a droplet digital PCR (ddPCR) was successfully developed to simultaneously detect the P. infestans CesA3 gene irrespective of its CAA sensitivity status, and either the CAA wild-type sequence for position 1105 or that conferring the resistant allele G1105S. Using a drop-off in ratio of positive droplets for either the wild-type or G1105S alleles relative to those positive for the general P. infestans CesA3 it was also possible to demonstrate that using the assay other potential alterations known to confer resistance that may occur at this position (e.g. G1105V) can be detected. The assay was validated using multiple sources of P. infestans DNA, including FTA cards. ConclusionsThe assay developed will allow for the accurate and sensitive detection of CAA resistance conferred by alterations at amino acid position 1105 in the CesA3 gene in P. infestans. The capacity to use the assay with multiple DNA sources and potential to adapt the drop-off approach to different ddPCR platforms will ensure its applicability to the wider P. infestans community in monitoring the continued use of CAA-based fungicides for P. infestans management.

microbiology↗

bioRxiv2025

Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While cytoplasmic macromolecular crowding affects multiple cellular processes in single cells, its regulation in living animals remains poorly understood. Using genetically encoded multimeric nanoparticles for in vivo rheology, we discovered that C. elegans tissues maintain distinct cytoplasmic biophysical properties that differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling cytoplasmic macromolecular diffusion: ribosome concentration, a known regulator of cytoplasmic crowding, works in concert with a previously unknown function for the giant KASH protein ANC-1 scaffolding the endoplasmic reticulum. These findings reveal mechanisms by which tissues establish and maintain distinct cytoplasmic biophysical properties, with implications for understanding cellular organization across species. One-Sentence SummaryLiving tissues maintain unique intracellular biophysical properties under the control of cytoplasmic constraints and crowding.

biophysics↗

bioRxiv2025

RhCMV Expands CCR5 Memory T Cells and promotes SIV reservoir seeding in the Gut Mucosa

Cytomegalovirus (CMV) is a prevalent {beta}-herpesvirus that persists asymptomatically in immunocompetent hosts. In people with HIV-1 (PWH), CMV is associated with persistence of the HIV-1 reservoir and particular inflammatory related co-morbidities. The true causative role of CMV in HIV-associated pathologies remains unclear given that nearly all PWH are coinfected with CMV. In this study, we examined acute phase SIV dynamics in cohorts of rhesus macaques that were seropositive or -negative for rhesus CMV (RhCMV). We observed expansion of CCR5+ target CD4+ T cells in gut and lymph nodes (LN) that existed naturally in RhCMV-seropositive animals, the majority of which did not react to RhCMV lysate. These cells expressed high levels of the chemokine receptor CXCR3 and a ligand for this receptor, CXCL9, was systemically elevated in RhCMV-seropositive animals. RhCMV+ RMs also exhibited higher peak SIV viremia. CCR5 target memory CD4 T cells in the gut of RhCMV+ RMs were maintained during acute SIV and this was associated with greater seeding of SIV DNA in the intestine. Overall, our data suggests the ability of RhCMV to regulate chemotactic axes that direct lymphocyte trafficking and promote seeding of SIV in a diverse, polyclonal pool of memory CD4+ T cells.

immunology↗

bioRxiv2024

Genomic epidemiology of emerging terbinafine-resistant Trichophyton indotineae

Dermatophyte skin infections affect around a quarter of the worlds population and are a growing public health concern due to increasing incidence of novel species causing severe infections that are resistant to antifungal treatments. Trichophyton species cause the greatest burden of dermatophytosis worldwide, with the T. mentagrophytes species complex being particularly associated with the emergence of new aggressive infections. One emerging species, T. indotineae (originally T. mentagrophytes genotype VIII) is notable for the extensive nature of the often inflammatory infection, its clinical resistance to terbinafine antifungal treatment, and its rapid global spread. To better understand the epidemiology of this disease, we sourced isolates from severe cases of dermatophytosis in the United Kingdom, Ireland, France, Canada and India for the period 2018-2023, including the type strain from Japan. We used whole-genome sequencing to confirm 90 isolates were T. indotineae, and antifungal susceptibility testing indicated that over half of these (62%) were resistant to terbinafine (MIC [≥]1 mg/L). Pairwise genetic distances showed very high identity with only 147 (1-414) SNPs separating isolates that were nested within a monophyletic phylogeny, supporting a single evolutionary origin of T. indotineae. That no clear geographic clustering of isolates was observed confirms the rapid transcontinental spread of T. indotineae from its likely centre of diversity in Asia. Genome-wide analyses identified multiple non-synonymous SNPs in SQLE (ERG1), the squalene epoxidase target of terbinafine, that were associated with terbinafine in vitro resistance [≥]1 mg/L. However, five isolates exhibited high MIC values without SQLE mutations, suggesting the presence of alternative resistance mechanisms. Our findings highlight the importance of better genomic surveillance to understand and manage this severe and rapidly emerging terbinafine-resistant dermatophyte.

genomics↗

bioRxiv2024

Comparative analysis of rhesus macaque and human placental organoids highlights evolutionary differences in placentation

Throughout evolution, the placenta has diversified in structure and cellular composition while maintaining its essential role in supporting fetal development. Trophoblasts, key cells responsible for nutrient exchange and immune modulation, are a conserved feature of all placentas. Although primate placentas share broad morphological similarities, species-specific differences in gene expression remain poorly characterized, largely due to the lack of tractable in vitro models. To address this gap, we developed rhesus macaque placental organoids representing both trophoblast and maternal-derived decidual cell types and compared them with human placental organoids. Using integrated single-cell and single-nucleus RNA sequencing, we identified both shared and species-specific transcriptional programs across corresponding trophoblast lineages. We further reconstructed lineage trajectories leading to multinucleated syncytiotrophoblast and invasive extravillous trophoblast populations, revealing conserved differentiation pathways alongside divergent gene expression signatures. This work establishes new in vitro models of the nonhuman primate placenta and defines the molecular distinctions between human and rhesus trophoblasts, offering insights into the evolutionary adaptations underlying placental development.

developmental biology↗

bioRxiv2024

Climatic fluctuations modulate eco-evolutionary processes associated with pathogen dynamics and continue to fuel bacterial spot epidemics in tomato

Recurring outbreaks caused by endemic pathogens continue to pose problems in managing plant, wildlife, livestock and human health. Understanding how these outbreaks unfold and what drives the variability in disease epidemics across space and time is less understood, especially in the agricultural settings. In this study, we investigated the contribution of pathogen genetic diversity, climatic variation and their interaction towards disease dynamics, with an integrative approach grounded on multitype, high resolution sequencing data and analysis techniques. This investigation was carried out for bacterial spot disease epidemic by surveying tomato fields for bacterial pathogen (Xanthomonas perforans) across southeastern US over a span of three years. The strength of epidemic severity varied across space and time in the agricultural fields. Disease severity was positively associated with strain diversity, and was linked to environmental fluctuations, specifically, large variation and extreme changes in certain climatic factors. Strain-resolved metagenomics approach revealed that co-existence of multiple pathogen lineages was common in the individual fields, although accompanied by differential lineage dynamics. The co-occurring lineages displayed environmentally dependent fitness contributions. By tracing allelic frequencies in pathogen populations across temporal scales, we find evidence for asynchronous allele cycling across seasons, hinting at the presence of adaptive single-nucleotide polymorphisms (SNPs) being polymorphic in space in response to seasonality. Despite this pathogen heterogeneity, we identified positively selected loci under parallel evolution, which may explain the nature of selection pressures experienced by the pathogen. While single pathogen lineage is assumed to dominate the host in the agricultural settings, our findings challenge this notion by demonstrating genetic diversity in the pathogen population observed within a single field and linking it to the disease dynamics. Our results explain the role of pathogen genetic diversity, climate-dependent compositional dynamics, and differential fitness contributions in dictating the variability of disease epidemics in the agricultural settings. Such findings will be invaluable for building predictive models in disease epidemiology. Our high-resolution combinatorial approach exploiting high- resolution sequence data, metadata types and analysis tools, is general enough to finely investigate disease epidemics at large scales in diverse case-studies concerning plant, animal and human health.

microbiology↗

bioRxiv2024

Natural variation in tetrapyrrole biosynthetic enzymes and their regulation modify the chlorophyll mutant Oy1-N1989

Tetrapyrroles are macrocyclic compounds present in and required for all life on Earth. Mutants with defects in tetrapyrrole pathway enzymes can be used to uncover natural variation in this pathway and study pathway regulation. We report here the effects of the Oy1-N1989/+ mutation, a semi-dominant allele of subunit I in the Mg-chelatase enzyme with reduced chlorophyll biosynthesis, on global gene expression and morphological traits. Coordinate regulation of the tetrapyrrole pathway was observed as transcriptional feedback regulation of genes in the tetrapyrrole pathway in Oy1-N1898/+ mutants. Natural variation in the wild-type allele at oy1 modulated the severity of the impact of Oy1-N1989/+ on gene expression. We also studied the effects of previously identified cis-acting expression variation at oy1 in wild-type plants. The seventy transcripts encoding biosynthetic enzymes in the tetrapyrrole pathway exhibited similar transcriptional co-regulation in response to cis-variation in oy1 transcript accumulation in wild-type plants as observed in the RNAseq of Oy1-N1989/+ mutants. This demonstrated that the coordinate regulation of the pathway also occurs during physiologically-relevant variation in OY1 abundance. Cis variants at seven tetrapyrrole pathway genes were linked to variation in chlorophyll accumulation in Oy1-N1989/+ mutant or wild-type plants. Analysis of trans-acting transcriptional variation by eGWAS detected multiple transcriptional hotspots, which disproportionately affected the expression of a subset of tetrapyrrole pathway genes, indicating that some genes are repeated targets of transcriptional regulation. The trans-regulatory hotspots coordinately regulated this pathway and may work to limit the accumulation of phototoxic intermediates. Author SummaryThe mutant allele and interactions with the genetic background determine the severity of mutant phenotypes. We used Oy1-N1989/+ mutants, affecting Mg-chelatase subunit I, to identify natural variants in the tetrapyrrole pathway affecting transcriptional regulation and variation in mutant traits. By integrating GWAS and transcriptomics, we identified similar feedback regulation of porphyrin and chlorophyll metabolism in mutants as well as in wild-type plants affected by natural variation. This is the first demonstration of natural variation affecting transcriptional feedback in the tetrapyrrole pathway under normal conditions. Using GWAS as a regulatory discovery tool, we identified unexplored regulatory mechanisms in a pathway that is critical for energy capture from the sun and chemical energy utilization in all living things and multiple human diseases.

plant biology↗

bioRxiv2024

Identification of ABHD6 as a regulator of lysophosphatidylserines in the mammalian liver and kidneys

Lysophosphatidylserine (lyso-PS) is a potent hormone-like signaling lysophospholipid, which regulates many facets of mammalian biology and dysregulation in its metabolism is associated with several human neurological and autoimmune diseases. Despite the physiological importance and causal relation with human pathophysiology, little is known about the metabolism of lyso-PS in tissues other than the nervous and immune systems. To address this problem, here, we attempted to identify one (or more) lipase(s) capable of degrading lyso-PS in different mammalian tissues. We found that the membrane proteomic fraction of most mammalian tissues possess lyso-PS lipase activity, yet interestingly, the only bona fide lyso-PS lipase ABHD12 displays this enzymatic activity and has control over lyso-PS metabolism only in the mammalian brain. Using an in vitro inhibitor screen against membrane proteomic fractions of different tissues, we find that another lipase from the metabolic serine hydrolase family, ABHD6, is a putative lyso-PS lipase in the mouse liver and kidney. Finally, using pharmacological tools, we validate the lyso-PS lipase activity of ABHD6 in vivo, and functionally designate this enzyme as a major lyso-PS lipase in primary hepatocytes, and the mammalian liver and kidneys. HIGHLIGHTSO_LILyso-PS lipase enzymatic activity is present in the membrane proteomic fraction of most mammalian tissues C_LIO_LIAn in vitro inhibitor screen identifies ABHD6 as a putative lyso-PS lipase in the liver and kidney C_LIO_LIABHD6 functions as a lyso-PS lipase in primary hepatocytes C_LIO_LIABHD6 also performs lyso-PS lipase activity in vivo and regulates lyso-PS levels in the liver and kidney C_LI

biochemistry↗

bioRxiv2024

The chromatin-associated 53BP1 ortholog, HSR-9, regulates recombinational repair and X chromosome segregation in the Caenorhabditis elegans germ line

53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the Caenorhabditis elegans ortholog of 53BP1, HSR-9, associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the X chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule-kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double strand breaks into COSA-1-marked crossovers. Additionally, HSR-9 plays a role in meiotic X chromosome segregation under conditions where X chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior. Article SummaryWhile 53BP1 is known for its crucial role in DNA damage signaling and repair in somatic cells, its role in meiosis is not well understood. Li, Hariri, et al., show that C. elegans 53BP1 not only functions in meiotic recombination and checkpoint signaling but also regulates the transmission of sex chromosomes when meiosis is perturbed. These results highlight the importance of 53BP1 in C. elegans meiosis and suggest that 53BP1 has both conserved and organism-specific functions.

genetics↗

bioRxiv2024

Within-host adaptation of a foliar pathogen, Xanthomonas, on pepper in presence of quantitative resistance and ozone stress

O_LIThe evolving threat of new pathogen variants in the face of global environmental changes poses a risk to the plant health and can impact the efficacy of resistance-based disease management. C_LIO_LIHere, we studied short-term eco-evolutionary response of the pathogen, Xanthomonas perforans, on quantitative resistant and susceptible pepper during a single growing season in open-top chambers under the influence of elevated Ozone (O3). C_LIO_LIWe observed increased disease severity, accompanied by higher variation on resistant cultivar under elevated O3, with no apparent change on the susceptible cultivar. This altered resistance response under elevated O3 is linked to altered eco-evolutionary dynamics of pathogen. While a single pathogen genotype remained prevalent on susceptible cultivar, resistant cultivar supported heterogenous pathogen population, with the evidence of short- term evolutionary modifications seeded by de novo parallel mutations. Altered O3 levels led to strain turnover on resistant cultivar with higher within-host polymorphism containing higher proportion of random de novo mutations lacking parallelism. C_LIO_LIPopulation heterogeneity is a mechanism of pathogen adaptation in response to the stressors. While parallel mutations in response to quantitative resistance may provide clues to predicting long-term pathogen evolution, high proportion of transient mutations suggest less predictable pathogen evolution under climatic alterations. C_LI

evolutionary biology↗

bioRxiv2024

Rhesus Cytomegalovirus-encoded Fcγ-binding glycoproteins facilitate viral evasion from IgG-mediated humoral immunity

Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFc{gamma}Rs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine the in vivo function of vFc{gamma}Rs in animal hosts closely related to humans, we identified and characterized vFc{gamma}Rs encoded by rhesus CMV (RhCMV). We demonstrate that Rh05, Rh152/151 and Rh173 represent the complete set of RhCMV vFc{gamma}Rs, each displaying functional similarities to their respective HCMV orthologs with respect to antagonizing host Fc{gamma}R activation in vitro. When RhCMV-naive rhesus macaques were infected with vFc{gamma}R-deleted RhCMV, peak plasma viremia levels and anti-RhCMV antibody responses were comparable to wildtype infections. However, the duration of plasma viremia was significantly shortened in immunocompetent, but not in CD4+ T cell-depleted animals. Since vFc{gamma}Rs were not required for superinfection, we conclude that vFc{gamma}Rs delay control by virus-specific adaptive immune responses, particularly antibodies, during primary infection.

microbiology↗

bioRxiv2024

Functional brain imaging predicts population-level visits to urban spaces

Urbanization is increasing around the world, and urban development strategies focusing on sustainability and the welfare of urban residents are needed. In response to this need, the field of neurourbanism has emerged, which leverages research on the human brain to understand and predict the influence of urban environments. For example, studying brain regions involved in reward processing and value-based decision making, such as the ventromedial prefrontal cortex (vmPFC), may help us understand how people interact with and navigate through urban environments. In this study, we aimed to ascertain whether neural activity within the vmPFC can predict population-level visits around the urban spaces of a city - in our case, Lisbon, Portugal. We used the density of photographs taken around Lisbon as a proxy measure of these visits. To do this, we created a stimulus set featuring 160 images of Lisbon sourced from the social media platform, Flickr. Then, study participants in the U.S. who had never visited Lisbon, viewed these images while we recorded their brain activity. We found that in our sample, activity in the vmPFC predicted the density of photographs taken around Lisbon, and hence, the population-level visits. Our research highlights the crucial role of the brain, especially reward-related brain regions, in shaping human behavior within urban environments. By shedding light on the neural mechanisms underlying urban behavior in humans, our research opens exciting possibilities for the future of urban planning. With this knowledge, policymakers and urban planners can potentially design cities that can promote well-being, social interaction, and sustainable living.

neuroscience↗

bioRxiv2024

Transcriptional profiling during infection of potato NLRs and Phytophthora infestans effectors using cDNA enrichment sequencing

An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions. Often, pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes. In this study, we compare two sequencing techniques, RNAseq and enrichment sequencing (RenSeq and PenSeq) of cDNA, to investigate gene expression patterns in the doubled monoploid potato (DM) infected with the late blight pathogen Phytophthora infestans. Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages. Throughout the infection time course, cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes. For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq, a high level of concordance in expression profiles is observed, indicative of at least semi-quantitative gene expression representation following enrichment.

molecular biology↗

bioRxiv2023

Comprehensive Codon Usage Analysis Across Diverse Plant Lineages

The variation of codon usage patterns in response to the evolution of organisms is an intriguing question to answer. The purpose of this study was to investigate the relevance of the evolutionary events of vascularization and seed production with the codon usage patterns in different plant lineages. We found that the optimal codons of non-vascular lineages generally end with GC, whereas those of the vascular lineages end with AU. Correspondence analysis and model-based clustering showed that the evolution of the codon usage pattern follows the evolutionary event of the vascularization more precisely than that of the seed production. The dinucleotides CpG and TpA were under-represented in all the lineages, whereas the dinucleotide TpG was found over-represented in all the lineages, except algae. Evolutionary-related lineages showed similar codon pair bias (CPB). The dinucleotide CpA showed a similar representation as those of its parent codon pairs. Although natural selection predominates over mutational pressure in determining the codon usage bias (CUB), the relative influence of mutational pressure is higher in the non-vascular lineages than in the vascular lineages.

evolutionary biology↗

bioRxiv2023

Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8 hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8 hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8 TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.

molecular biology↗