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Accumulating evidence demonstrates that CD8(+) T cells contribute to protection from severe dengue virus (DENV) disease and vaccine efficacy. Nevertheless, molecular programs associated with DENV-specific CD8(+) T cell subsets have not been defined. Here, we studied the transcriptomic profiles of human DENV-specific CD8(+) T cells isolated after stimulation with DENV epitopes from donors who had been infected with DENV multiple times and would therefore be expected to have significant levels of adaptive immunity. We found that DENV-specific CD8(+) T cells mainly consisted of effector memory subsets, namely CD45RA(−)CCR7(−) effector memory (Tem) and CD45RA(+)CCR7(−) effector memory re-expressing CD45RA (Temra) cells, which enacted specific gene expression profiles upon stimulation with cognate antigens. DENV-specific CD8(+) T cell subsets in general, and Temra cells in particular, were fully activated and polyfunctional, yet associated with relatively narrow transcriptional responses. Furthermore, we found that DENV-specific CD8(+) Tem and Temra cells showed some unique T cell receptor features in terms of overlap and variable (V) gene usage. This study provides a transcriptomic definition of DENV-specific activated human CD8(+) T cell subsets and defines a benchmark profile that vaccine-specific responses could aim to reproduce.

The increased incidence of whooping cough worldwide suggests that current vaccination against Bordetella pertussis infection has limitations in quality and duration of protection. The resurgence of infection has been linked to the introduction of acellular vaccines (aP), which have an improved safety profile compared with the previously used whole-cell (wP) vaccines. To determine immunological differences between aP and wP priming in infancy, we performed a systems approach of the immune response to booster vaccination. Transcriptomic, proteomic, cytometric, and serologic profiling revealed multiple shared immune responses with different kinetics across cohorts, including an increase of blood monocyte frequencies and strong antigen-specific IgG responses. Additionally, we found a prominent subset of aP-primed individuals (30%) with a strong differential signature, including higher levels of expression for CCL3, NFKBIA, and ICAM1. Contrary to the wP individuals, this subset displayed increased PT-specific IgE responses after boost and higher antigen-specific IgG4 and IgG3 antibodies against FHA and FIM2/3 at baseline and after boost. Overall, the results show that, while broad immune response patterns to Tdap boost overlap between aP- and wP-primed individuals, a subset of aP-primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.

Whole-cell inactivated vaccine against Bordetella pertussis (wP) was substituted in many countries by an acellular subunit vaccine (aP) to reduce side effects. Recent epidemiological studies have shown that aP vaccination in infancy induces less durable immunity than wP vaccination. To determine immunological differences associated with aP vs. wP priming, we performed system-level profiling of the immune response in adults primed with aP vs. wP vaccine in infancy following the Tdap booster vaccination as a surrogate to antigen encounter in vivo. Shared immune responses across cohorts were identified, including an increase of the blood monocyte frequency on day 1, and strong antigen-specific IgG response seven days after boost. Comparing aP and wP primed individuals, we found a subset of aP-primed individuals with higher levels of expression for several genes including CCL3 on day 3 and NFKBIA and ICAM1 on day 7 post immunization. These observations were supported by increased CCL3 concentrations in plasma of aP primed individuals. Contrary to the wP individuals, the CCL3-high aP subset presented boosted PT-specific IgE responses. Furthermore, higher antigen specific IgG4 and IgG3 antibodies against specific vaccine antigens at baseline and post boost of aP individuals was observed, suggesting a long term maintained difference in the IgG subtype response. Overall our findings demonstrate that, while broad immune response patterns to Tdap boost overlap between aP and wP primed individuals, a subset of aP primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.

et al., Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype–phenotype relationships1,2. Here we present a human ‘all-by-all’ reference interactome map of human binary protein interactions, or ‘HuRI’. With approximately 53,000 protein–protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein–protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes., This work was primarily supported by the National Institutes of Health (NIH) National Human Genome Research Institute (NHGRI) grant U41HG001715 (M.V., F.P.R., D.E.H., M.A.C., G.D.B. and J.T.) with additional support from NIH grants P50HG004233 (M.V. and F.P.R.), U01HL098166 (M.V.), U01HG007690 (M.V.), R01GM109199 (M.A.C.), Canadian Institute for Health Research (CIHR) Foundation Grants (F.P.R. and J. Rak), the Canada Excellence Research Chairs Program (F.P.R.) and an American Heart Association grant 15CVGPS23430000 (M.V.). D.-K.K. was supported by a Banting Postdoctoral Fellowship through the Natural Sciences and Engineering Research Council (NSERC) of Canada and by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2017R1A6A3A03004385). C. Pons was supported by a Ramon Cajal fellowship (RYC-2017-22959). G.M.S. was supported by NIH Training Grant T32CA009361. M.V. is a Chercheurv Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

Global insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human “all-by-all” binary reference interactome map, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artefacts and thus ignored in data acquisition and analysis, are the result of true biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell:monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be revisited.

Tuberculosis (TB) is a major infectious disease worldwide, and is associated with several challenges for control and eradication. First, more accurate diagnostic tools that better represent the spectrum of infection states are required; in particular, identify the latent TB infected individuals with high risk of developing active TB. Second, we need to better understand, from a mechanistic point of view, why the immune system is unsuccessful in some cases for control and elimination of the pathogen. Host transcriptomics is a powerful approach to identify both diagnostic and mechanistic immune signatures of diseases. We have recently reported that optimal study design for these two purposes should be guided by different sets of criteria. Here, based on already published transcriptomics signatures of tuberculosis, we further develop these guidelines and identify additional factors to consider for obtaining diagnostic vs. mechanistic signatures in terms of cohorts, samples, data generation and analysis. Diagnostic studies should aim to identify small disease signatures with high discriminatory power across all affected populations, and against similar pathologies to TB. Specific focus should be made on improving the diagnosis of infected individuals at risk of developing active disease. Conversely, mechanistic studies should focus on tissues biopsies, immune relevant cell subsets, state of the art transcriptomic techniques and bioinformatics tools to understand the biological meaning of identified gene signatures that could facilitate therapeutic interventions. Finally, investigators should ensure their data are made publicly available along with complete annotations to facilitate metadata and cross-study analyses.

In the mid-1990s, whole-cell pertussis (wP) vaccines were associated with local and systemic adverse events that prompted their replacement with acellular pertussis (aP) vaccines in many high-income countries. In the past decade, rates of pertussis disease have increased in children receiving only aP vaccines. We compared the immune responses to aP boosters in individuals who received their initial doses with either wP or aP vaccines using activation-induced marker (AIM) assays. Specifically, we examined pertussis-specific memory CD4+ T cell responses ex vivo, highlighting a type 2/Th2 versus type 1/Th1 and Th17 differential polarization as a function of childhood vaccination. Remarkably, after a contemporary aP booster, cells from donors originally primed with aP were (a) associated with increased IL-4, IL-5, IL-13, IL-9, and TGF-β and decreased IFN-γ and IL-17 production, (b) defective in their ex vivo capacity to expand memory cells, and (c) less capable of proliferating in vitro. These differences appeared to be T cell specific, since equivalent increases of antibody titers and plasmablasts after aP boost were seen in both groups. In conclusion, our data suggest that there are long-lasting effects and differences in polarization and proliferation of T cell responses in adults originally vaccinated with aP compared with those that initially received wP, despite repeated acellular boosters.

The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56+ TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56+ TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens., Memory T cells are essential for combating recurring infection by promoting prompt and effective immune responses. Here the authors show, via system biology approaches, that human CD4 memory T cells contains a CD45RA-rexpressing pool that can be further subsetted by the expression of GPR56 for distinct functionalities.

In the mid-1990s vaccine-related side effects prompted the replacement of the whole Pertussis (wP) by a new acellular Pertussis vaccine (aP). Unexpectedly, whooping cough cases have recently increased, and the switch to the aP vaccine suspected to underlie this rise in morbidity, despite the use of routine boosters. Here we compared the immune responses to aP boosters in children who received their initial doses with either wP or aP vaccines. The use of ex vivo AIM assays highlighted a Th2 vs Th1/Th17 differential polarization of PT-specific memory CD4+ T cells as a function of childhood vaccination. The nature of the differences was further investigated for memory subset composition, activation, exhaustion marker expression, and transcriptomic profiles. Remarkably, donors originally primed with aP were defective in their ex vivo capacity to expand memory cells following a booster aP immunization and less capable to proliferate in vitro in response to PT epitopes. By contrast antibody responses are boosted in both aP and wP cohorts following IgG subclass distribution. Most recently, we have assessed early immune responses followed aP boosting for the aP and wP cohorts using longitudinal transcriptomics data from PBMC. We have identified several modules of genes that are co-expressed; using cell frequency data from CyTOF we infer modules correlated with specific cell types. Furthermore, we identified certain modules that show differential expression profiles between the two cohorts and are mostly enriched in immune related functions. In conclusion, our data suggest that the original priming after birth with aP and wP vaccines induce different T cell phenotypes associated with long-term T cell polarization.

Computational protein design methods can complement experimental screening and selection techniques by predicting libraries of low-energy sequences compatible with a desired structure and function. Incorporating backbone flexibility in computational design allows conformational adjustments that should broaden the range of predicted low-energy sequences. Here, we evaluate computational predictions of sequence libraries from different protocols for modeling backbone flexibility using the complex between the therapeutic antibody Herceptin and its target human epidermal growth factor receptor 2 (HER2) as a model system. Within the program RosettaDesign, three methods are compared: The first two use ensembles of structures generated by Monte Carlo protocols for near-native conformational sampling: kinematic closure (KIC) and backrub, and the third method uses snapshots from molecular dynamics (MD) simulations. KIC or backrub methods were better able to identify the amino acid residues experimentally observed by phage display in the Herceptin–HER2 interface than MD snapshots, which generated much larger conformational and sequence diversity. KIC and backrub, as well as fixed backbone simulations, captured the key mutation Asp98Trp in Herceptin, which leads to a further threefold affinity improvement of the already subnanomolar parental Herceptin-HER2 interface. Modeling subtle backbone conformational changes may assist in the design of sequence libraries for improving the affinity of antibody–antigen interfaces and could be suitable for other protein complexes for which structural information is available.

The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen-binding site can significantly enlarge the repertoire. Among the six hyper-variable complementarity determining regions (CDRs) that comprise the binding site, the CDR H3 loop is particularly flexible. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi-constraint computational design strategy, along with the Rosetta energy function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen-bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by pre-organizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity.

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.

Protein metal binding sites in the pre-bound (apo) state, and their rearrangements upon metal binding were not analyzed previously at a database scale. Such a study may provide valuable information for metal binding site prediction and design. A high resolution, nonredundant dataset of 210 metal binding sites was created, containing all available representatives of apo-holo pairs for the most populated metals in the PDB. More than 40% of the sites underwent rearrangements upon metal binding. In 30 cases rearrangements involved the backbone. The tendency for side-chain rearrangement inversely correlates with the number of first-shell residues. Analysis of side-chain reorientations as a result of metal binding showed that in 95% of the rigid-backbone binding sites at most one side chain moved. Thus, in general, part of the first coordination shell is already in place in the pre-bound form. The frequencies of side-chain reorientation directly correlated with metal ligand flexibility and solvent accessibility in the apo state.

Dengue virus (DENV) has spread through most tropical and subtropical areas of the world and represents a serious public health problem. The control of DENV infections has been hindered by lack of effective therapeutics or vaccines. Nevertheless, a better understanding of the immune system both at steady state and following DENV infections may reveal new strategies for eliciting and improving anti-DENV immunity. T cells provide protective immunity against various viral infections, and recent findings demonstrate that a subset of CD4 T cells, designated effector memory re-expressing CD45RA T (Temra) cells, expand following DENV infections and may play an important role in protecting the host from severe dengue diseases. In this study, we report that CD4 Temra cells display unique gene expression profiles characterized by the upregulation of cytotoxic molecules such as perforin, granzyme B and GPR56, as well as transcription factors including T-bet, Eomes, Runx3 and Hobit, which direct T cell differentiation, function and tissue residency. Furthermore, our results indicate that CD4 Temra cells are heterogeneous and can be largely divided into two sub-populations, which exhibited differential expression of various effector molecules and transcriptional regulators. Finally, using high-dimensional mass cytometry, we further dissected the heterogeneity of DENV-specific CD4 Temra cells. Over 30 molecules were analyzed simultaneously, resulting in the identification of novel subsets within the CD4 Temra population. Overall, our findings reveal immune signatures of CD4 Temra cells in healthy and DENV-infected individuals and provide new insights into the antiviral immunity against DENV and potentially other emerging pathogens.

Analysis of the spatial arrangement of protein and water atoms that form polar interactions with ribose has been performed for a structurally non-redundant dataset of ATP, ADP and FAD-protein complexes. The 26 ligand-protein structures were separated into two groups corresponding to the most populated furanose ring conformations (N and S-domains). Four conserved positions were found for S-domain protein-ligand complexes and five for N-domain complexes. Multiple protein folds and secondary structural elements were represented at a single conserved position. The following novel points were revealed: (i) Two complementary positions sometimes combine to describe a putative atomic spatial location for a specific conserved binding spot. (ii) More than one third of the interactions scored were water-mediated. Thus, conserved spatial positions rich in water atoms are a significant feature of ribose-protein complexes.

Yeast Sec18p and its mammalian orthologue N- ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two β-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.

SummaryAlternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide “microexons” display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.