Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5′ untranslated region (UTR). Differential 5′ UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5′ UTR lengthened by over 100 bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.

Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve expressed antibody (Ab) transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted amplification by 5’ rapid amplification of cDNA ends (RACE), combined with single molecule, real-time sequencing to generate highly accurate (>Q40, 99.99%) IG heavy chain transcripts. FLAIRR-seq was benchmarked by comparing IG heavy chain variable (IGHV), diversity (IGHD), and joining (IGHJ) gene usage, complementarity-determining region 3 (CDR3) length, and somatic hypermutation to matched datasets generated with standard 5’ RACE AIRR-seq and full-length isoform sequencing. Together these data demonstrate robust, unbiased FLAIRR-seq performance using RNA samples derived from peripheral blood mononuclear cells, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving novel IG heavy chain constant (IGHC) gene features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class-switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk expressed Ab repertoires to date.

S1: Unaltered tumor burden in colons of ACKR2-/-ApcMin/+ mice. S2: Delayed onset of tumor development in the small intestine of ACKR2-/-ApcMin/+ mice. S3. Decreased inflammatory markers in the distal intestine tumors of ACKR2-/-ApcMin/+ mice. S4. Elevated mast cell infiltration into microadenomas in ACKR2-/-ApcMin/+ mice. S5. Identification of BMMC. S6. Relative expression levels of chemokine receptors in BMMC. S7. Dinitrophenyl (DNP) induced calcium release in BMMC. S8. Analysis of CCR2 and CCR5 expression in immune cells from ACKR2-/- mice. S9. Uptake and MHC-1 mediated presentation of peptide antigens by mast cells. S10. Mast cell infiltration into ACKR2-/- tumors is not dependent on T cells. Supplemental Table 1: List of primers used for mouse genotyping. Supplemental Table 2: List of mouse Real Time PCR primer sequences.

The Fc receptor for IgM, FcMR, is unusual in that it is preferentially expressed by cells of the adaptive immune system. It is, moreover, the only constitutively expressed Fc receptor on human T cells. Efforts to decipher the normal functions of FcMR have been complicated by species-specific expression patterns in lymphocytes from mice (B cells) versus humans (B, NK, and T cells). In human cells, FcMR cell-surface expression has been reported to be low at baseline ex vivo, with one suggested contribution being ligand-induced internalization by serum IgM. Indeed, preincubation overnight in IgM-free culture medium is recommended for studies of FcMR because surface display is increased under these conditions. We investigated FcMR display on human lymphocytes in PBMCs and found that, surprisingly, cell-surface FcMR was unaffected by IgM abundance and was instead downregulated in high-cell density cultures by a yet undefined mechanism. We further found that ex vivo processing of whole blood decreased surface FcMR, supporting the idea that FcMR expression is likely to be greater on circulating lymphocytes than previously appreciated. Collectively, these findings prompt new predictions of where and when FcMR might be available for functional interactions in vivo.

Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.

Surface modified microspheres have been leveraged as a useful way to immobilize antigen for serological studies. The use of carboxyl modified microspheres for this purpose is well-established, but commonly associated with technical challenges. Streptavidin modified microspheres require little technical expertise and thus address some of the shortcomings of carboxyl microspheres. An additional feature of streptavidin microspheres is the use of mono-biotinylated proteins, which contain a single biotinylation motif at the C-terminus. However, the relative performance of streptavidin and carboxyl microspheres is unknown. Here, we performed a head-to-head comparison of streptavidin and carboxyl microspheres. We compared antigen binding, orientation, and staining quality and found that both microspheres perform similarly based on these defined parameters. We also evaluated the utility of streptavidin microspheres bound to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD), to reliably detect RBD-specific IgG1, IgG3, and IgA1 produced in individuals recently immunized with Pfizer/BioNTech mRNA coronavirus disease (COVID) vaccine as 'proof-of-concept'. We provide evidence that each of the antibody targets are detectable in serum using RBD-coated microspheres, Ig-specific 'detector' monoclonal antibodies (mAbs), and flow cytometry. We found that cross-reactivity of the detector mAbs can be minimized by antibody titration to improve differentiation between IgG1 and IgG3. We also coated streptavidin microspheres with SARS-CoV-2 delta variant RBD to determine if the streptavidin microsphere approach revealed any differences in binding of immune serum antibodies to wild-type (Wuhan) versus variant RBD (Delta). Overall, our results show that streptavidin microspheres loaded with mono-biotinylated antigen is a robust alternative to chemically cross-linking antigen to carboxyl microspheres for use in serological assays.

Alphaviruses are arthropod-borne RNA viruses which can cause either mild to severe febrile arthritis which may persist for months, or encephalitis which can lead to death or lifelong cognitive impairments. The non-assembly molecular role(s), functions, and protein–protein interactions of the alphavirus capsid proteins have been largely overlooked. Here we detail the use of a BioID2 biotin ligase system to identify the protein–protein interactions of the Sindbis virus capsid protein. These efforts led to the discovery of a series of novel host–pathogen interactions, including the identification of an interaction between the alphaviral capsid protein and the host IRAK1 protein. Importantly, this capsid–IRAK1 interaction is conserved across multiple alphavirus species, including arthritogenic alphaviruses SINV, Ross River virus, and Chikungunya virus; and encephalitic alphaviruses Eastern Equine Encephalitis virus, and Venezuelan Equine Encephalitis virus. The impact of the capsid–IRAK1 interaction was evaluated using a robust set of cellular model systems, leading to the realization that the alphaviral capsid protein specifically inhibits IRAK1-dependent signaling. This inhibition represents a means by which alphaviruses may evade innate immune detection and activation prior to viral gene expression. Altogether, these data identify novel capsid protein–protein interactions, establish the capsid–IRAK1 interaction as a common alphavirus host–pathogen interface, and delineate the molecular consequences of the capsid–IRAK1 interaction on IRAK1-dependent signaling.

Monophosphoryl lipid A (MPL®) is the first non-alum vaccine adjuvant to achieve widespread clinical and market acceptance, a remarkable achievement given that it is manufactured from a Salmonella enterica endotoxin. To understand how MPL® successfully balanced the dual mandate of vaccine design—low reactogenicity with high efficacy—clinical- and research-grade MPL was evaluated in human and mouse cell systems. Stimulatory dose response curves revealed that most preparations of MPL are much more active in mouse than in human cell systems, and that the limited efficacy observed in human cells correlated with TLR4 inhibitory activity that resulted in a partial agonist profile. Further analysis of the major components of MPL® adjuvant prepared synthetically identified two structural variants that functioned as competitive antagonists of human TLR4. A partial agonist profile could be recapitulated and manipulated by spiking synthetic agonists with synthetic antagonists to achieve a broad dose range over which TLR4 stimulation could be constrained below a desired threshold. This report thus identifies mixed agonist–antagonist activity as an additional mechanism by which MPL® adjuvant is detoxified, relative to its parental LPS, to render it safe for use in prophylactic vaccines.

Phylogenomic studies have so far mostly relied on genome skimming or target sequence capture, which suffer from representation bias and can fail to resolve relationships even with hundreds of loci. Here, we explored the potential of phylogenetic informativeness and tree confidence analyses to interpret phylogenomic datasets. We studied Cucurbitaceae because their small genome size allows cost-efficient genome skimming, and many relationships in the family remain controversial, preventing inferences on the evolution of characters such as sexual system or floral morphology. Genome skimming and PCR allowed us to retrieve the plastome, 57 single copy nuclear genes, and the nuclear ribosomal ITS from 29 species representing all but one tribe of Cucurbitaceae. Node support analyses revealed few inter-locus conflicts but a pervasive lack of phylogenetic signal among plastid loci, suggesting a fast divergence of Cucurbitaceae tribes. Data filtering based on phylogenetic informativeness and risk of homoplasy clarified tribe-level relationships, which support two independent evolutions of fringed petals in the family. Our study illustrates how formal analysis of phylogenomic data can increase our understanding of past diversification processes. Our data and results will facilitate the design of well-sampled phylogenomic studies in Cucurbitaceae and related families.

LPS delays neutrophil apoptosis by a process generally assumed to involve cell-intrinsic TLR4 signaling. However, neutrophil survival responses to LPS have been reported to be monocyte-dependent, which would indicate more complexity than is currently appreciated. We compared the survival responses of conventionally purified vs highly purified neutrophils to confirm or refute the need for secondary cell-types and to identify the cellular or molecular mechanisms involved. Direct stimulation of TLR4 failed to extend the survival of highly purified neutrophils, but survival activity was retained in less pure neutrophil preparations containing low numbers of eosinophils, monocytes, platelets and CD3 + lymphocytes. Sequential depletions identified monocytes as the only cell type required. Transfer of culture supernatants after lipid A-conditioning revealed that purified monocytes were sufficient for production of nearly all of the survival activity observed in mixed populations. The survival factors secreted upon TLR4 stimulation remain unidentified, but were not correlated with IL-1β, IL-6 or TNF-α nor could survival activity be inhibited by Ab blockade of IL-8 or of several other candidate factors other than endogenously produced GM-CSF, which was responsible for about one-tenth of the survival activity present in conditioned supernatants. These observations confirm that ex vivo neutrophil survival responses to TLR4 agonists are not cell intrinsic and involve potentially novel factors secreted by TLR4-stimulated monocytes.

Abstract The presence of mast cells in some human colorectal cancers is a positive prognostic factor, but the basis for this association is incompletely understood. Here, we found that mice with a heterozygous mutation in the adenomatous polyposis coli gene (ApcMin/+) displayed reduced intestinal tumor burdens and increased survival in a chemokine decoy receptor, ACKR2-null background, which led to discovery of a critical role for mast cells in tumor defense. ACKR2–/–ApcMin/+ tumors showed increased infiltration of mast cells, their survival advantage was lost in mast cell–deficient ACKR2–/–SA–/–ApcMin/+ mice as the tumors grew rapidly, and adoptive transfer of mast cells restored control of tumor growth. Mast cells from ACKR2–/– mice showed elevated CCR2 and CCR5 expression and were also efficient in antigen presentation and activation of CD8+ T cells. Mast cell–derived leukotriene B4 (LTB4) was found to be required for CD8+ T lymphocyte recruitment, as mice lacking the LTB4 receptor (ACKR2–/–BLT1–/–ApcMin/+) were highly susceptible to intestinal tumor-induced mortality. Taken together, these data demonstrate that chemokine-mediated recruitment of mast cells is essential for initiating LTB4/BLT1-regulated CD8+ T-cell homing and generation of effective antitumor immunity against intestinal tumors. We speculate that the pathway reported here underlies the positive prognostic significance of mast cells in selected human tumors. Cancer Immunol Res; 6(3); 332–47. ©2018 AACR.

Abstract A neutrophil culture method recapitulates their phenotypic plasticity and may accelerate development of drugs that target pathological subsets in airway disease.

Development of non-infectious subunit vaccines is hampered by a slow pipeline of new adjuvants to replace or enhance alum in part because expectations of safety are high. Transient vaccine side effects are not clinical priorities because they cause no lasting harm and vaccine development has appropriately been focused on avoidance of serious adverse events. As a result, surprisingly little is known about the extent to which side effects caused by a vaccine's reactogencicity are predictive of successful immunization outcomes. Recent clinical studies of pertussis and human papillomavirus vaccines adjuvanted with alum or the TLR4 agonist monophosphoryl lipid A can be used to advance understanding of the relationship between vaccine side effects and immunization outcomes.

Abstract Pseudomonas aeruginosa infection in cystic fibrosis (CF) lung disease causes airway neutrophilia and hyperinflammation without being cleared effectively. We evaluated the immunostimulatory activities of lipid A variants synthesized by P. aeruginosa (PA) exclusively in CF patients to determine if they correlate with CF disease severity and progression. One third of the PA isolates from CF patients with late severe stage express a unique hepta–acylated lipid A; hepta-1855 (m/z=1855) isoform. In primary cell cultures, we found that hepta-1855 functioned as a potent TLR4 agonist by priming neutrophil respiratory burst and stimulating strong chemokine response (interleukin-8 or IL-8) from monocytes and neutrophils. Hepta-1855 also had a potent survival effect on neutrophils. However, it was less efficient in inducing neutrophil granule exocytosis and was also less potent in triggering pro-inflammatory TNF-α response from monocytes. A precursor hexa-1616, found in hepta-1855 containing LPS mixtures, did not have direct inflammatory activity on neutrophils but induced moderate IL-8 response. Together, our data suggest a potential contribution of hepta-1855 to late CF stage associated excessive inflammatory burden by recruiting neutrophils via IL-8 and promoting their maintenance through its survival effect. Being a chemoattractant stimulus, hexa-1616, may serve as an accomplice to hepta-1855. Moreover, the relative inefficiency of hepta-1855 in triggering degranulation may partly explain the persistence of PA in CF disease in spite of neutrophil dominated inflammation.

Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) lung disease causes airway neutrophilia and hyperinflammation without effective bacterial clearance. We evaluated the immunostimulatory activities of lipid A, the membrane anchor of LPS, isolated from mutants of PA that synthesize structural variants, present in the airways of patients with CF, to determine if they correlate with disease severity and progression. In a subset of patients with a severe late stage of CF disease, a unique hepta-acylated lipid A, hepta-1855, is synthesized. In primary human cell cultures, we found that hepta-1855 functioned as a potent TLR4 agonist by priming neutrophil respiratory burst and stimulating strong IL-8 from monocytes and neutrophils. hepta-1855 also had a potent survival effect on neutrophils. However, it was less efficient in stimulating neutrophil granule exocytosis and also less potent in triggering proinflammatory TNF-α response from monocytes. In PA isolates that do not synthesize hepta-1855, a distinct CF-specific adaptation favors synthesis of a penta-1447 and hexa-1685 LPS mixture. We found that penta-1447 lacked immunostimulatory activity but interfered with inflammatory IL-8 synthesis in response to hexa-1685. Together, these observations suggest a potential contribution of hepta-1855 to maintenance of the inflammatory burden in late-stage CF by recruiting neutrophils via IL-8 and promoting their survival, an effect presumably amplified by the absence of penta-1447. Moreover, the relative inefficiency of hepta-1855 in triggering neutrophil degranulation may partly explain the persistence of PA in CF disease, despite extensive airway neutrophilia.

Background: At a global scale, the temperate zone is highly fragmented both between and within hemispheres. This paper aims to investigate how the world's disjunct temperate zones have been colonised by the pan-temperate plant group Convolvuleae, sampling 148 of the c. 225 known species. We specifically determine the number and timing of amphitropical and transoceanic disjunctions, investigate the extent to which disjunctions in Convolvuleae are spatio-temporally congruent with those in other temperate plant groups and determine the impact of long-distance dispersal events on diversification rates.Results: Eight major disjunctions are observed in Convolvuleae: two Northern Hemisphere, two Southern Hemisphere and four amphitropical. Diversity in the Southern Hemisphere is largely the result of a single colonisation of Africa 3.1-6.4 Ma, and subsequent dispersals from Africa to both Australasia and South America. Speciation rates within this monophyletic, largely Southern Hemisphere group (1.38 species Myr(-1)) are found to be over twice those of the tribe as a whole (0.64 species Myr(-1)). Increased speciation rates are also observed in Calystegia (1.65 species Myr(-1)).Conclusions: The Convolvuleae has colonised every continent of the world with a temperate biome in c. 18 Myr and eight major range disjunctions underlie this broad distribution. In keeping with other temperate lineages exhibiting disjunct distributions, long-distance dispersal is inferred as the main process explaining the patterns observed although for one American-Eurasian disjunction we cannot exclude vicariance. The colonisation of the temperate zones of the three southern continents within the last c. 4 Myr is likely to have stimulated high rates of diversification recovered in this group, with lineage accumulation rates comparable to those reported for adaptive radiations.

A global revision of Convolvulus L. is presented, Calystegia R.Br. being excluded on pragmatic grounds. One hundred and ninety species are recognised with the greatest diversity in the Irano-Turanian region. All recognised species are described and the majority are illustrated. Distribution details, keys to species identification and taxonomic notes are provided. Four new species, Convolvulus austroafricanus J.R.I.Wood & R.W.Scotland, sp. nov., Convolvulus iranicus J.R.I.Wood & R.W.Scotland, sp. nov., Convolvulus peninsularis J.R.I.Wood & R.W.Scotland, sp. nov. and Convolvulus xanthopotamicus J.R.I.Wood & R.W.Scotland, sp. nov., one new subspecies Convolvulus chinensis subsp. triangularis J.R.I.Wood & R.W.Scotland, subsp. nov., and two new varieties Convolvulus equitans var. lindheimeri J.RI.Wood & R.W.Scotland, var. nov., Convolvulus glomeratus var. sachalitarum J.R.I.Wood & R.W.Scotland, var. nov. are described. Convolvulus incisodentatus J.R.I.Wood & R.W.Scotland, nom. nov., is provided as a replacement name for the illegitimate Convolvulus incisus Choisy. Several species treated as synonyms of other species in recent publications are reinstated including C. chinensis Ker-Gawl., C. spinifer M.Popov., C. randii Rendle and C. aschersonii Engl Ten taxa are given new status and recognised at new ranks: Convolvulus namaquensis (Schltr. ex. A.Meeuse) J.R.I.Wood & R.W.Scotland, stat. nov., Convolvulus hermanniae subsp. erosus (Desr.) J.R.I.Wood & R.W.Scotland, stat nov., Convolvulus crenatifolius subsp. montevidensis (Spreng.) J.R.I.Wood & R.W.Scotland, stat nov., Convolvulus fruticulosus subsp. glandulosus (Webb) J.R.I.Wood & R.W.Scotland, stat nov., Convolvulus capituliferus subsp. foliaceus (Verdc.) J.R.I.Wood & R.W.Scotland, stat nov., Convolvulus hystrix subsp. ruspolii (Dammer ex Hallier f.) J.R.I.Wood & R.W.Scotland, stat. nov., Convolvulus hystrix subsp. inermis (Chiov.) J.R.I.Wood & R.W.Scotland, stat. nov., Convolvulus rottlerianus subsp. stocksii (Boiss.) J.R.I.Wood & R.W.Scodand, comb. et stat. nov., Convolvulus calvertii subsp. ruprechtii (Boiss.) J.R.I.Wood & R.W.Scodand, stat. nov., Convolvulus cephalopodus subsp. bushiricus (Bornm.) J.R.I.Wood & R.W.Scodand, stat. nov. The status of various infraspecific taxa is clarified and numerous taxa are lectotypified. This account represents a new initiative in terms of taxonomic monography, being an attempt to bring together the global approach of the traditional monograph with the more pragmatic and identification-focussed approach of most current floras while at the same time being informed by insights from molecular systematics.

Linnaeosicyos amara is a little known cucurbit endemic to the Caribbean island of Hispaniola. The species possesses conspicuously fringed petals which are known in just four other cucurbit genera and hypothesised to play a role in pollinator attraction. A population of flowering L amara plants was observed for a total of 78 h, which revealed that Sphingid moths are the primary pollinators. Floral scent was dominated by the terpenoid (E)-beta-ocimene. The elaborate petal fringes almost doubled the apparent diameter of L. amara flowers and the maximal extension of petal fringes was found to coincide with the peak visitation period of hawk-moths, with withering starting soon after. We conclude that petal fringes likely play a role in the visual attraction of hawk-moths by exploiting their preference for large flowers with deeply-divided petal margins, whilst limiting energy costs to the plant. (C) 2015 Elsevier GmbH. All rights reserved.