Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information.

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systematizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

Parasite manipulation of host phenotypes inferred from transcriptional analyses in a trematode-amphipod system.

Manipulation of host phenotypes by parasites is hypothesized to be an adaptive strategy enhancing parasite transmission across hosts and generations. Characterizing the molecular mechanisms of manipulation is important to advance our understanding of host-parasite coevolution. The trematode (Levinseniella byrdi) is known to alter the colour and behaviour of its amphipod host (Orchestia grillus) presumably increasing predation of amphipods which enhances trematode transmission through its life cycle. We sampled 24 infected and 24 uninfected amphipods from a salt marsh in Massachusetts to perform differential gene expression analysis. In addition, we constructed novel genomic tools for O. grillus including a de novo genome and transcriptome. We discovered that trematode infection results in upregulation of amphipod transcripts associated with pigmentation and detection of external stimuli, and downregulation of multiple amphipod transcripts implicated in invertebrate immune responses, such as vacuolar ATPase genes. We hypothesize that suppression of immune genes and the altered expression of genes associated with coloration and behaviour may allow the trematode to persist in the amphipod and engage in further biochemical manipulation that promotes transmission. The genomic tools and transcriptomic analyses reported provide new opportunities to discover how parasites alter diverse pathways underlying host phenotypic changes in natural populations.

Continuity and Change in U.S. Children's Family Composition, 1968-2017.

We document changes in U.S. children's family household composition from 1968 to 2017 with regard to the number and types of kin that children lived with and the frequency of family members' household entrances and departures. Data are from the U.S. Panel Study of Income Dynamics (N = 30,412). Children experienced three decades of increasing instability and diversification in household membership, arriving at a state of "stable complexity" in the most recent decade. Stable complexity is distinguished by a decline in the number of coresident parents; a higher number of stepparents, grandparents, and other relatives in children's households; and less turnover in household membership compared with prior decades, including fewer sibling departures. College-educated households with children were consistently the most stable and least diverse. On several dimensions, household composition has become increasingly similar for non-Hispanic Black and White children. Children in Hispanic households are distinct in having larger family sizes and more expected household entrances and departures by coresident kin.

Global change effects on plant communities are magnified by time and the number of global change factors imposed.

Global change drivers (GCDs) are expected to alter community structure and consequently, the services that ecosystems provide. Yet, few experimental investigations have examined effects of GCDs on plant community structure across multiple ecosystem types, and those that do exist present conflicting patterns. In an unprecedented global synthesis of over 100 experiments that manipulated factors linked to GCDs, we show that herbaceous plant community responses depend on experimental manipulation length and number of factors manipulated. We found that plant communities are fairly resistant to experimentally manipulated GCDs in the short term (<10 y). In contrast, long-term (≥10 y) experiments show increasing community divergence of treatments from control conditions. Surprisingly, these community responses occurred with similar frequency across the GCD types manipulated in our database. However, community responses were more common when 3 or more GCDs were simultaneously manipulated, suggesting the emergence of additive or synergistic effects of multiple drivers, particularly over long time periods. In half of the cases, GCD manipulations caused a difference in community composition without a corresponding species richness difference, indicating that species reordering or replacement is an important mechanism of community responses to GCDs and should be given greater consideration when examining consequences of GCDs for the biodiversity-ecosystem function relationship. Human activities are currently driving unparalleled global changes worldwide. Our analyses provide the most comprehensive evidence to date that these human activities may have widespread impacts on plant community composition globally, which will increase in frequency over time and be greater in areas where communities face multiple GCDs simultaneously.

Single-cell transcriptomics reveals the effect of PD-L1/TGF-ß blockade on the tumor microenvironment.

BACKGROUND: The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-ß has been shown to contribute to T cell exclusion, and anti-TGF-ß improves anti-PD-L1 efficacy in vivo. However, TGF-ß inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-ß blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-ß blockade in the tumor microenvironment should be further elucidated. RESULTS: We used single-cell RNA sequencing in a mouse model to characterize the transcriptomic effects of PD-L1 and/or TGF-ß blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in macrophages, and downregulation of extracellular matrix genes in fibroblasts. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1. CONCLUSIONS: Taken together, our data could be leveraged translationally to complement or find alternatives to anti-PD-L1 plus anti-TGF-ß combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

Determinants of community compositional change are equally affected by global change.

Global change is impacting plant community composition, but the mechanisms underlying these changes are unclear. Using a dataset of 58 global change experiments, we tested the five fundamental mechanisms of community change: changes in evenness and richness, reordering, species gains and losses. We found 71% of communities were impacted by global change treatments, and 88% of communities that were exposed to two or more global change drivers were impacted. Further, all mechanisms of change were equally likely to be affected by global change treatments-species losses and changes in richness were just as common as species gains and reordering. We also found no evidence of a progression of community changes, for example, reordering and changes in evenness did not precede species gains and losses. We demonstrate that all processes underlying plant community composition changes are equally affected by treatments and often occur simultaneously, necessitating a wholistic approach to quantifying community changes.

Not All Nitrogen Is Created Equal: Differential Effects of Nitrate and Ammonium Enrichment in Coastal Wetlands.

Excess reactive nitrogen (N) flows from agricultural, suburban, and urban systems to coasts, where it causes eutrophication. Coastal wetlands take up some of this N, thereby ameliorating the impacts on nearshore waters. Although the consequences of N on coastal wetlands have been extensively studied, the effect of the specific form of N is not often considered. Both oxidized N forms (nitrate, NO3-) and reduced forms (ammonium, NH4+) can relieve nutrient limitation and increase primary production. However, unlike NH4+, NO3- can also be used as an electron acceptor for microbial respiration. We present results demonstrating that, in salt marshes, microbes use NO3- to support organic matter decomposition and primary production is less stimulated than when enriched with reduced N. Understanding how different forms of N mediate the balance between primary production and decomposition is essential for managing coastal wetlands as N enrichment and sea level rise continue to assail our coasts.

Ambient changes exceed treatment effects on plant species abundance in global change experiments.

The responses of species to environmental changes will determine future community composition and ecosystem function. Many syntheses of global change experiments examine the magnitude of treatment effect sizes, but we lack an understanding of how plant responses to treatments compare to ongoing changes in the unmanipulated (ambient or background) system. We used a database of long-term global change studies manipulating CO2 , nutrients, water, and temperature to answer three questions: (a) How do changes in plant species abundance in ambient plots relate to those in treated plots? (b) How does the magnitude of ambient change in species-level abundance over time relate to responsiveness to global change treatments? (c) Does the direction of species-level responses to global change treatments differ from the direction of ambient change? We estimated temporal trends in plant abundance for 791 plant species in ambient and treated plots across 16 long-term global change experiments yielding 2,116 experiment-species-treatment combinations. Surprisingly, for most species (57%) the magnitude of ambient change was greater than the magnitude of treatment effects. However, the direction of ambient change, whether a species was increasing or decreasing in abundance under ambient conditions, had no bearing on the direction of treatment effects. Although ambient communities are inherently dynamic, there is now widespread evidence that anthropogenic drivers are directionally altering plant communities in many ecosystems. Thus, global change treatment effects must be interpreted in the context of plant species trajectories that are likely driven by ongoing environmental changes.

A distinct evolution of the T-cell repertoire categorizes treatment refractory gastrointestinal acute graft-versus-host disease.

Steroid refractory gastrointestinal (GI) acute graft-versus-host disease (aGVHD) is a major cause of mortality in hematopoietic stem cell transplantation (HCT) without immune markers to establish a diagnosis or guide therapy. We found that T-cell receptor ß (TCRß) complementarity-determining region 3 repertoire sequencing reveals patterns that could eventually serve as a disease biomarker of T-cell alloreactivity in aGVHD. We identified T-cell clones in GI biopsies in a heterogeneous group of 15 allogeneic HCT patients with GI aGVHD symptoms. Seven steroid-refractory aGVHD patients showed a more conserved TCRß clonal structure between different biopsy sites in the GI tract than 8 primary therapy-responsive patients. Tracking GI clones identified longitudinally at endoscopy in the blood also revealed an increased clonal expansion in patients with steroid-refractory disease. Immune repertoire sequencing-based methods could enable a novel personalized way to guide diagnosis and therapy in diseases where T-cell activity is a major determinant.

A fiddler crab reduces plant growth in its expanded range.

Species across the planet are shifting or expanding their ranges because of climate change. These are climate migrants. Although climate migrants are well documented, their impacts on recipient ecosystems are not. Climate migrants that are also ecosystem engineers (species that modify or create habitats) will likely have profound effects on ecosystems. The Atlantic marsh fiddler crab, Minuca pugnax, is a burrowing crab that recently expanded its range into the northeastern United States. In its historical range, M. pugnax enhances the aboveground growth of the cordgrass Spartina alterniflora, a plant critical to marsh persistence. In a control-impact study, however, we found that Spartina aboveground biomass was 40% lower when M. pugnax was present. Thus, the positive effect of M. pugnax on Spartina aboveground biomass flipped to a negative one in its expanded range. Spartina belowground biomass was also 30% lower on average when crabs were present, a finding consistent with what is seen in the historical range. These impacts on Spartina are likely due to burrowing by M. pugnax. Benthic microalgae was, on average, 45% lower when crabs were present. Fiddler crabs eat benthic microalgae, and these results suggest that fiddler crabs can control algal biomass via grazing. Because fiddler crabs reduced the biomass of foundational primary producers in its expanded range, our results imply that M. pugnax can influence other saltmarsh functions such as carbon storage and accretion as they expand north. Most strikingly, our results suggest that as species expand or shift their range with climate change, not only can they have profound impacts in their new ranges but those impacts can be the inverse of what is seen in their historical ranges.

Sequence features that drive human promoter function and tissue specificity.

Promoters are important regulatory elements that contain the necessary sequence features for cells to initiate transcription. To functionally characterize a large set of human promoters, we measured the transcriptional activities of 4575 putative promoters across eight cell lines using transient transfection reporter assays. In parallel, we measured gene expression in the same cell lines and observed a significant correlation between promoter activity and endogenous gene expression (r = 0.43). As transient transfection assays directly measure the promoting effect of a defined fragment of DNA sequence, decoupled from epigenetic, chromatin, or long-range regulatory effects, we sought to predict whether a promoter was active using sequence features alone. CG dinucleotide content was highly predictive of ubiquitous promoter activity, necessitating the separation of promoters into two groups: high CG promoters, mostly ubiquitously active, and low CG promoters, mostly cell line-specific. Computational models trained on the binding potential of transcriptional factor (TF) binding motifs could predict promoter activities in both high and low CG groups: average area under the receiver operating characteristic curve (AUC) of the models was 91% and exceeded the AUC of CG content by an average of 23%. Known relationships, for example, between HNF4A and hepatocytes, were recapitulated in the corresponding cell lines, in this case the liver-derived cell line HepG2. Half of the associations between tissue-specific TFs and cell line-specific promoters were new. Our study underscores the importance of collecting functional information from complementary assays and conditions to understand biology in a systematic framework.

Predicting antibody binders and generating synthetic antibodies using deep learning.

The antibody drug field has continually sought improvements to methods for candidate discovery and engineering. Historically, most such methods have been laboratory-based, but informatics methods have recently started to make an impact. Deep learning, a subfield of machine learning, is rapidly gaining prominence in the biomedical research. Recent advances in microfluidics technologies and next-generation sequencing have not only revolutionized therapeutic antibody discovery, but also contributed to a vast amount of antibody repertoire sequencing data, providing opportunities for deep learning-based applications. Previously, we used microfluidics, yeast display, and deep sequencing to generate a panel of binder and non-binder antibody sequences to the cancer immunotherapy targets PD-1 and CTLA-4. Here we encoded the antibody light and heavy chain complementarity-determining regions (CDR3s) into antibody images, then built and trained convolutional neural network models to classify binders and non-binders. To improve model interpretability, we performed in silico mutagenesis to identify CDR3 residues that were important for binder classification. We further built generative deep learning models using generative adversarial network models to produce synthetic antibodies against PD-1 and CTLA-4. Our models generated variable length CDR3 sequences that resemble real sequences. Overall, our study demonstrates that deep learning methods can be leveraged to mine and learn patterns in antibody sequences, offering insights into antibody engineering, optimization, and discovery.

GMP Manufacturing and IND-Enabling Studies of a Recombinant Hyperimmune Globulin Targeting SARS-CoV-2.

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Arterial injury in total knee arthroplasty.

Arterial complications associated with knee arthroplasty are relatively rare, although probably underreported, complications of knee arthroplasty that carry a risk of significant morbidity. Thorough preoperative assessment and close liaison with a vascular surgeon, combined with an appreciation of common anatomical variants or distorted anatomy, may help prevent both thromboembolic and direct injuries from occurring. Clinical features of arterial complications following knee arthroplasty may vary significantly from acute hemorrhage or ischemia in the immediate postoperative period to chronic pain and swelling presenting even months following the procedure. There is potential for diagnostic confusion and delay that may adversely affect outcome. Early diagnosis along with vascular surgical review and intervention is key to successful management.

Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR.

IN VITRO: affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we performed affinity maturation using a yeast-based single-chain variable fragment (scFv) expression system to compare two mutagenesis methods: random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions (CDRs). We applied both methods of mutagenesis to four human antibodies against well-known immuno-oncology target proteins. Detailed sequence analysis showed an even mutational distribution across the entire length of the scFv for the error-prone PCR method and an almost exclusive targeting of the CDRs for the combinatorial method. Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved scFv affinity with similar efficiency. When a subset of the affinity-matured antibodies was expressed as full-length immunoglobulin, the measured affinity constants were mostly comparable to those of the respective scFv, but the full-length antibodies were inferior to their scFv counterparts for one of the targets. Furthermore, we found that improved affinity for the full-length antibody did not always translate into enhanced binding to cell-surface expressed antigen or improved immune checkpoint blocking ability, suggesting that screening with full-length antibody or antigen-binding fragment formats might be advantageous and the subject of a future study.

Recovery of the salt marsh periwinkle (Littoraria irrorata) 9 years after the Deepwater Horizon oil spill: Size matters.

Prior studies indicated salt marsh periwinkles (Littoraria irrorata) were strongly impacted in heavily oiled marshes for at least 5 years following the Deepwater Horizon oil spill. Here, we detail longer-term effects and recovery over nine years. Our analysis found that neither density nor population size structure recovered at heavily oiled sites where snails were smaller and variability in size structure and density was increased. Total aboveground live plant biomass and stem density remained lower over time in heavily oiled marshes, and we speculate that the resulting more open canopy stimulated benthic microalgal production contributing to high spring periwinkle densities or that the lower stem density reduced the ability of subadults and small adults to escape predation. Our data indicate that periwinkle population recovery may take one to two decades after the oil spill at moderately oiled and heavily oiled sites, respectively.

Massively parallel interrogation and mining of natively paired human TCRαß repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics.

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.

Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries.

To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism.