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1.  Distinct Roles of Cytoskeletal Components in Immunological Synapse Formation and Directed Secretion 
A hallmark of CD4+ T cell activation and immunological synapse (IS) formation is the migration of the microtubule organization center (MTOC) and associated organelles toward the APC. In this study, we found that when murine CD4+ T cells were treated with a microtubule-destabilizing agent (vinblastine) after the formation of IS, the MTOC dispersed and all of the major cellular organelles moved away from the IS. Cytokines were no longer directed toward the synapse but were randomly secreted in quantities similar to those seen in synaptic secretion. However, if the actin cytoskeleton was disrupted at the same time with cytochalasin D, the organelles did not shift away from the IS. These findings suggest that there is a complex interplay between the microtubules and actin cytoskeleton, where microtubules are important for directing particular cytokines into the synapse but they are not involved in the amount of cytokines that are produced for at least 1 h after IS formation. Additionally, we found that they play a critical role in mobilizing organelles to reorient toward the synapse during T cell activation and in stabilizing organelles against the force that is generated through actin polymerization so that they move toward the APC. These findings show that there is a complex interplay between these major cytoskeletal components during synapse formation and maintenance.
doi:10.4049/jimmunol.1402175
PMCID: PMC4610857  PMID: 26392461
2.  Single-cell systems level analysis of human Toll-Like-Receptor activation defines a chemokine signature in Systemic Lupus Erythematosus 
Background
Activation of Toll-Like Receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in Systemic Lupus Erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has previously not been described.
Objective
To characterize TLR activation across multiple immune cell subsets and individuals, with the goal of establishing a reference framework against which to compare pathological processes.
Methods
Peripheral whole blood samples were stimulated with TLR ligands, and analyzed by mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied seventeen healthy volunteer donors and eight newly diagnosed untreated SLE patients.
Results
Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of NK and T cells selectively induced NF-κB in response to TLR2 ligands. CD14hi monocytes exhibited the most polyfunctional cytokine expression patterns, with over 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared amongst a group of healthy donors, with minimal intra- and inter- individual variability. Furthermore, autoimmune disease altered baseline cytokine production, as newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.
Conclusion
Mass cytometry analysis defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in inflammatory disease, such as SLE.
doi:10.1016/j.jaci.2015.04.008
PMCID: PMC4640970  PMID: 26037552
Mass cytometry; Toll-Like-Receptors; systemic lupus erythematosus; inflammation; monocytes; MCP1
3.  Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol 
Nature immunology  2016;17(7):844-850.
Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor–CD3 complex (TCR). Here we show that cholesterol sulfate (CS), a naturally occurring analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. Biochemical studies show that CS disrupted TCR multimers, apparently by displacing cholesterol, known to bind TCRβ. Moreover, CS-deficient mice displayed a heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation.
doi:10.1038/ni.3462
PMCID: PMC4916016  PMID: 27213689
5.  New approaches to understanding the immune response to vaccination and infection 
Vaccine  2015;33(40):5271-5281.
Summary
The immune system is a network of specialized cell types and tissues that communicates via cytokines and direct contact, to orchestrate specific types of defensive responses. Until recently, we could only study immune responses in a piecemeal, highly focused fashion, on major components like antibodies to the pathogen. But recent advances in technology and in our understanding of the many components of the system, innate and adaptive, have made possible a broader approach, where both the multiple responding cells and cytokines in the blood are measured. This systems immunology approach to a vaccine response or an infection gives us a more holistic picture of the different parts of the immune system that are mobilized and in turn, may allow us to better understand the mechanisms of such responses, as well as to predict vaccine efficacy in different populations well in advance of efficacy studies. Here we summarize the different technologies and methods and discuss how they can inform us about the differences between diseases and vaccines, and how they can greatly accelerate vaccine development.
doi:10.1016/j.vaccine.2015.06.117
PMCID: PMC4581990  PMID: 26232539
systems immunology; immune profiling; high-throughput methods; vaccinology; computational immunology; regularization; feature selection; elastic net; human immunology
6.  Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells 
T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.
doi:10.4049/jimmunol.1502031
PMCID: PMC5055199  PMID: 27655845
7.  Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals 
PLoS Pathogens  2016;12(10):e1005892.
Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.
Author Summary
Vaccination is one of the most successful medical interventions, but it loses its effectiveness in an older population that is of particular risk for infectious diseases. Shingles, caused by the reactivation of the chickenpox virus, is a prime example. Nearly every second individual has experienced shingles by the age of 80 years, and the shingles vaccine is only partially protective. Attempts to improve the vaccine response are mostly empiric. Vaccinations induce a rapid expansion of antigen-specific T cells with frequencies peaking after one to two weeks. Most expanded T cells die after the peak response, and only few T cells survive to provide protection from infection or, as in case of shingles, from reactivation of latent viruses. Most vaccine studies have focused on the early stages of the response; how T cells are activated and expand. Surprisingly, in our study with the shingle vaccine, T cell survival after the peak response was the major factor determining memory T cell frequencies. T cell attrition was increased with age, independent of genetic predisposition. Using systems biology tools we found several pathways involved in T cell division and DNA repair that could be targeted to improve T cell survival and thereby increase the effectiveness of vaccination.
doi:10.1371/journal.ppat.1005892
PMCID: PMC5072604  PMID: 27764254
8.  Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching 
eLife  null;5:e16578.
Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state.
DOI: http://dx.doi.org/10.7554/eLife.16578.001
eLife digest
The human immune system comprises cells and processes that protect the body against infection and disease. B cells are immune cells that once activated produce antibodies, or proteins that help identify and neutralize infectious microbes and diseased host cells.
Antibodies fall into one of ten different classes, and each class has a different, specialized role. Certain antibody classes are responsible for eradicating viruses, while others recruit and help activate additional cells of the immune system.
B cells multiply quickly once they are activated. During this proliferation process, dividing B cells can switch from making one class of antibody to another. As such, a single activated B cell can yield a group of related B cells that produce distinct classes of antibodies. Although much has been learned about antibody class switching and its role in generating a diverse set of antibodies, the process of creating different antibody classes in humans remains unknown.
Horns, Vollmers et al. now reveal how antibodies of every class are created in living humans. By developing a way to reconstruct the B cell proliferation process and thereby trace the lineage of individual B cells, the occurrence of class switching events could be measured and mapped. This approach revealed that most antibodies are produced via a single dominant pathway that involves first switching through one of two antibody classes.
Horns, Vollmers et al. also determined that closely related B cells, which were recently born through division of a common ancestor, often switched to the same class. The shared fate is likely explained by the existence of similar conditions inside each cell, which are inherited during cell division and direct switching toward a particular class. All together, these new findings lay a foundation for developing techniques to direct antibody class switching in ways that support the immune system. Future work will aim to understand the conditions inside a cell that direct switching toward a particular class of antibody.
DOI: http://dx.doi.org/10.7554/eLife.16578.002
doi:10.7554/eLife.16578
PMCID: PMC4970870  PMID: 27481325
antibody; class switching; repertoire; cell decisions; Human
9.  Pregnancy Does Not Attenuate the Antibody or Plasmablast Response to Inactivated Influenza Vaccine 
The Journal of Infectious Diseases  2015;212(6):861-870.
Background. Inactivated influenza vaccine (IIV) is recommended during pregnancy to prevent influenza infection and its complications in pregnant women and their infants. However, the extent to which pregnancy modifies the antibody response to vaccination remains unclear, and prior studies have focused primarily on hemagglutinin inhibition (HI) titers. A more comprehensive understanding of how pregnancy modifies the humoral immune response to influenza vaccination will aid in maximizing vaccine efficacy.
Methods. Healthy pregnant women and control women were studied prior to, 7 days after, and 28 days after vaccination with IIV. HI titers, microneutralization (MN) titers, and the frequency of circulating plasmablasts were evaluated in pregnant versus control women.
Results. Pregnant women and control women mount similarly robust serologic immune responses to IIV, with no significant differences for any influenza strain in postvaccination geometric mean HI or MN titers. HI and MN titers correlate, though MN titers demonstrate more robust changes pre- versus postvaccination. The induction of circulating plasmablasts is increased in pregnant women versus controls (median fold-change 2.60 vs 1.49 [interquartile range, 0.94–7.53 vs 0.63–2.67]; P = .03).
Conclusions. Pregnant women do not have impaired humoral immune responses to IIV and may have increased circulating plasmablast production compared to control women.
doi:10.1093/infdis/jiv138
PMCID: PMC4548461  PMID: 25740957
hemagglutinin inhibition; influenza; plasmablast; pregnancy; vaccination; viral neutralization
10.  mir-181a-1/b-1 Modulates Tolerance through Opposing Activities in Selection and Peripheral T Cell Function 
Understanding the consequences of tuning T cell receptor (TCR) signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. Here we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive (DP) thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Together, these results demonstrate that tolerance can be modulated by miRNAs through the control of opposing activities in T cell selection and peripheral T cell function.
doi:10.4049/jimmunol.1401587
PMCID: PMC4763610  PMID: 26163591
11.  Mass cytometry analysis shows that a novel memory phenotype B cell is expanded in multiple myeloma 
Cancer immunology research  2015;3(6):650-660.
It would be very beneficial if the status of cancers could be determined from a blood specimen. However, peripheral blood leukocytes are very heterogeneous between individuals and thus high resolution technologies are likely required. We used cytometry by time-of-flight (CyTOF) and next generation sequencing to ask whether a plasma cell cancer (multiple myeloma) and related pre-cancerous states had any consistent effect on the peripheral blood mononuclear cell phenotypes of patients. Analysis of peripheral blood samples from 13 cancer patients, 9 pre-cancer patients, and 9 healthy individuals revealed significant differences in the frequencies of the T, B, and natural killer cell compartments. Most strikingly, we identified a novel B-cell population that normally accounts for 4.0±0.7% (mean±SD) of total B cells and is up to 13-fold expanded in multiple myeloma patients with active disease. This population expressed markers previously associated with both memory (CD27+) and naïve (CD24loCD38+) phenotypes. Single-cell immunoglobulin gene sequencing showed polyclonality, indicating that these cells are not precursors to the myeloma, and somatic mutations, a characteristic of memory cells. SYK, ERK, and p38 phosphorylation responses, and the fact that most of these cells expressed isotypes other than IgM or IgD, confirmed the memory character of this population, defining it as a novel type of memory B cells.
doi:10.1158/2326-6066.CIR-14-0236-T
PMCID: PMC4457663  PMID: 25711758
Multiple Myeloma; Tumor Immunology; B-Lymphocyte Biology; Immune Phenotyping; Peripheral Blood
12.  A population response analysis approach to assign class II HLA-epitope restrictions 
Identification of the specific HLA locus and allele presenting an epitope for recognition by specific T cell receptors (HLA restriction) is necessary to fully characterize the immune response to antigens. Experimental determination of HLA restriction is complex and technically challenging. As an alternative, the restricting HLA locus and allele can be inferred by genetic association, utilizing response data in an HLA typed population. However, simple odds ratio calculations can be problematic when dealing with large numbers of subjects and antigens and because the same epitope can be presented by multiple alleles (epitope promiscuity). Here, we develop a tool, denominated Restrictor Analysis Tool for Epitopes (RATE), to extract inferred restriction from HLA class II -typed epitope responses. This automated method infers HLA class II restriction from large datasets of T cell responses in HLA class II typed subjects by calculating Odds Ratios and relative frequencies from simple data tables. The program is validated by 1. Analyzing data of previously determined HLA restrictions. 2. Experimentally determining in selected individuals new HLA restrictions using HLA transfected cell lines 3. Predicting HLA restriction of particular peptides, and showing that corresponding HLA class II tetramers efficiently bind to epitope specific T cells. We further design a specific iterative algorithm to account for promiscuous recognition by calculation of Odds Ratio values for combinations of different HLA molecules while incorporating predicted HLA binding affinity. The RATE program streamlines the prediction of HLA class II restriction across multiple T cell epitopes and HLA types.
doi:10.4049/jimmunol.1403074
PMCID: PMC4458389  PMID: 25948811
HLA restriction; MHC; epitope; T cell; tetramer
13.  The Promised Land of Human Immunology 
Advances in technology and data analysis have made it possible to take a new look at human immunology. These advances run the gamut from systems biology approaches, which are likely in the vanguard of how we can start “to put the pieces together” of immune function, to a deeper understanding of specific diseases and vaccines and the immune repertoire. In our own experience, we have also found that asking simple questions about human immunity has often given us very surprising answers, causing a rethink of established dogma. Thus, we have developed a new perspective on the nature of the αβ TCR repertoire and also the likely role of T-cell repertoire (TCR) cross-reactivity in generating T memory independent of specific antigen interactions. These findings show that human immunology is not just a necessary step for “translating” basic immunology to treat diseases or develop better vaccines, but is also an important complement to the inbred mouse model.
doi:10.1101/sqb.2013.78.022905
PMCID: PMC4896736  PMID: 24638855
14.  Functional Development of the T Cell Receptor for Antigen 
For over three decades now, the T cell receptor (TCR) for antigen has not ceased to challenge the imaginations of cellular and molecular immunologists alike. T cell antigen recognition transcends every aspect of adaptive immunity: it shapes the T cell repertoire in the thymus and directs T cell-mediated effector functions in the periphery, where it is also central to the induction of peripheral tolerance. Yet, despite its central position, there remain many questions unresolved: how can one TCR be specific for one particular peptide-major histocompatibility complex (pMHC) ligand while also binding other pMHC ligands with an immunologically relevant affinity? And how can a T cell’s extreme specificity (alterations of single methyl groups in their ligand can abrogate a response) and sensitivity (single agonist ligands on a cell surface are sufficient to trigger a measurable response) emerge from TCR–ligand interactions that are so low in affinity? Solving these questions is intimately tied to a fundamental understanding of molecular recognition dynamics within the many different contexts of various T cell–antigen presenting cell (APC) contacts: from the thymic APCs that shape the TCR repertoire and guide functional differentiation of developing T cells to the peripheral APCs that support homeostasis and provoke antigen responses in naïve, effector, memory, and regulatory T cells. Here, we discuss our recent findings relating to T cell antigen recognition and how this leads to the thymic development of foreign-antigen-responsive αβT cells.
doi:10.1016/S1877-1173(10)92004-8
PMCID: PMC4887107  PMID: 20800817
15.  Clonal Deletion Prunes, but does not Eliminate Self-Specific αβ CD8+ T Lymphocytes 
Immunity  2015;42(5):929-941.
SUMMARY
It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. But here we found that self peptide-MHC specific CD8+ T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome encoded SMCY antigen, self-specific T cells exhibited only a three-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.
doi:10.1016/j.immuni.2015.05.001
PMCID: PMC4455602  PMID: 25992863
16.  High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry 
Mucosal immunology  2015;9(1):68-82.
In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus double-layered particles with a metal isotope and characterized B cells specific to the rotavirus VP6 major structural protein. We describe the heterogeneity of the intestinal B cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B cell subsets in the intestine and blood, and revealed that IgM+ memory B cells (MBCs) and naïve B cells were phenotypically related as were CD27− MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B cell subsets in immune donors, including naïve B cells, with phenotypes representative of the overall B cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.
doi:10.1038/mi.2015.36
PMCID: PMC4618273  PMID: 25899688
Human intestinal B cells; mass cytometry; rotavirus
17.  Distinct Patterns of B-Cell Activation and Priming by Natural Influenza Virus Infection Versus Inactivated Influenza Vaccination 
The Journal of Infectious Diseases  2014;211(7):1051-1059.
Background. The human B-cell response to natural influenza virus infection has not been extensively investigated at the polyclonal level.
Methods. The overall B-cell response of patients acutely infected with the 2009 pandemic influenza A(H1N1)pdm09 virus (A[H1N1]pdm09) was analyzed by determining the reactivity of plasmablast-derived polyclonal antibodies (PPAbs) to influenza proteins. Recipients of inactivated influenza vaccine containing the same A(H1N1)pdm09 strain were studied for comparison.
Results. During acute infection, robust plasmablast responses to the infecting virus were detected, characterized by a greater PPAb reactivity to the conserved influenza virus nuclear protein and to heterovariant and heterosubtypic hemagglutinins, in comparison to responses to the inactivated A(H1N1)pdm09 vaccine. In A(H1N1)pdm09 vaccinees, the presence of baseline serum neutralizing antibodies against A(H1N1)pdm09, suggesting previous exposure to natural A(H1N1)pdm09 infection, did not affect the plasmablast response to vaccination, whereas repeated immunization with inactivated A(H1N1)pdm09 vaccine resulted in significantly reduced vaccine-specific and cross-reactive PPAb responses.
Conclusions. Natural A(H1N1)pdm09 infection and inactivated A(H1N1)pdm09 vaccination result in very distinct patterns of B-cell activation and priming. These differences are likely to be associated with differences in protective immunity, especially cross-protection against heterovariant and heterosubtypic influenza virus strains.
doi:10.1093/infdis/jiu580
PMCID: PMC4366605  PMID: 25336731
influenza virus infection; influenza vaccine; B-cell response; antibody; plasmablast
18.  IgH sequences in common variable immune deficiency reveal altered B cell development and selection** 
Science translational medicine  2015;7(302):302ra135.
Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ∼1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity determining region 3 (CDR3). We observed decreased selection against antibodies with long CDR3 regions in memory repertoires and decreased V gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive both from decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. CVID patients also exhibited abnormal clonal expansion of unmutated B cells relative to controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B cell stage and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.
doi:10.1126/scitranslmed.aab1216
PMCID: PMC4584259  PMID: 26311730
19.  The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ Receptors 
Vaccine  2014;32(45):5989-5997.
Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or “split” viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors—specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus “splitting” inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.
doi:10.1016/j.vaccine.2014.07.115
PMCID: PMC4191649  PMID: 25203448
influenza; vaccine; Fcγ Receptors; immunology; mass cytometry
20.  Variation in the human immune system is largely driven by non-heritable influences 
Cell  2015;160(0):37-47.
SUMMARY
There is considerable heterogeneity in immunological parameters between individuals, but its sources are largely unknown. To assess the relative contribution of heritable versus non-heritable factors, we have performed a systems-level analysis of 210 healthy twins between 8–82 years of age. We measured 204 different parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated (> 50% of variance) and 58% almost completely determined (> 80% of variance) by non-heritable influences. In addition, some of these parameters become more variable with age, suggesting the cumulative influence of environmental exposure. Similarly, the serological responses to seasonal influenza vaccination are also determined largely by non-heritable factors, likely due to repeated exposure to different strains. Lastly, in MZ twins discordant for cytomegalovirus infection, over half of all parameters are affected. These results highlight the largely reactive and adaptive nature of the immune system in healthy individuals.
doi:10.1016/j.cell.2014.12.020
PMCID: PMC4302727  PMID: 25594173
21.  Cytomegalovirus infection improves immune responses to influenza 
Science translational medicine  2015;7(281):281ra43.
Cytomegalovirus (CMV) is a beta-herpes virus present in a latent form in most people worldwide. In immunosuppressed individuals, CMV can reactivate and cause serious clinical complications, but the effect of the latent state on healthy people remains elusive. We undertook a systems approach to understand the differences between seropositive and negative subjects and measured hundreds of immune system components from blood samples including cytokines and chemokines, immune cell phenotyping, gene expression, ex vivo cell responses to cytokine stimuli and the antibody response to seasonal influenza vaccination. As expected, we found decreased responses to vaccination and an overall down-regulation of immune components in aged individuals regardless of CMV serostatus. In contrast, CMV-infected young adults exhibited an overall up-regulation of immune components including enhanced antibody responses to influenza vaccination, increased CD8+ T cell sensitivity, and elevated levels of circulating IFN-γ compared to uninfected individuals. Experiments with young mice infected with murine CMV also showed significant protection from an influenza virus challenge compared with uninfected animals, although this effect declined with time. These data show that CMV and its murine equivalent can have a beneficial effect on the immune response of young, healthy individuals, which may explain the continued coexistence of CMV and mammals throughout their evolution.
doi:10.1126/scitranslmed.aaa2293
PMCID: PMC4505610  PMID: 25834109
22.  Distinct Roles of Cytoskeletal Components in Immunological Synapse Formation and Directed Secretion 
A hallmark of CD4+ T cell activation and immunological synapse (IS) formation is the migration of the microtubule organization center and associated organelles toward the APCs. In this study, we found that when murine CD4+ T cells were treated with a microtubule-destabilizing agent (vinblastine) after the formation of IS, the microtubule organization center dispersed and all of the major cellular organelles moved away from the IS. Cytokines were no longer directed toward the synapse but were randomly secreted in quantities similar to those seen in synaptic secretion. However, if the actin cytoskeleton was disrupted at the same time with cytochalasin D, the organelles did not shift away from the IS. These findings suggest that there is a complex interplay between the microtubules and actin cytoskeleton, where microtubules are important for directing particular cytokines into the synapse, but they are not involved in the amount of cytokines that are produced for at least 1 h after IS formation. In addition, we found that they play a critical role in mobilizing organelles to reorient toward the synapse during T cell activation and in stabilizing organelles against the force that is generated through actin polymerization so that they move toward the APCs. These findings show that there is a complex interplay between these major cytoskeletal components during synapse formation and maintenance.
doi:10.4049/jimmunol.1402175
PMCID: PMC4610857  PMID: 26392461
23.  Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements 
Cell host & microbe  2014;16(1):105-114.
Summary
B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual’s secreted antibody response to the vaccine and the expanded clones are enriched for those expressing influenza-specific mAbs. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.
doi:10.1016/j.chom.2014.05.013
PMCID: PMC4158033  PMID: 24981332
24.  Deconstructing the peptide-MHC specificity of T cell recognition 
Cell  2014;157(5):1073-1087.
Summary
In order to survey a universe of MHC-presented peptide antigens whose numbers greatly exceed the diversity of the T cell repertoire, T cell receptors (TCRs) are thought to be cross-reactive. However, the nature and extent of TCR cross-reactivity has not been conclusively measured experimentally. We developed a system to identify MHC-presented peptide ligands by combining TCR selection of highly diverse yeast-displayed peptide-MHC libraries with deep sequencing. While we identified hundreds of peptides reactive with each of five different mouse and human TCRs, the selected peptides possessed TCR recognition motifs that bore a close resemblance to their known antigens. This structural conservation of the TCR interaction surface allowed us to exploit deep sequencing information to computationally identify activating microbial and self-ligands for human autoimmune TCRs. The mechanistic basis of TCR cross-reactivity described here enables effective surveillance of diverse self and foreign antigens, but without necessitating degenerate recognition of non-homologous peptides.
doi:10.1016/j.cell.2014.03.047
PMCID: PMC4071348  PMID: 24855945
25.  Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells 
Nature biotechnology  2014;32(2):149-157.
Adaptive immune responses often begin with the formation of a molecular complex between a T cell receptor (TCR) and a peptide antigen bound to a major histocompatibility complex (MHC) molecule. These complexes are highly variable, however, due to the polymorphism of MHC genes, the random, inexact recombination of TCR gene segments and the vast array of possible self and pathogen peptide antigens. As a result, it has been very difficult to comprehensively study the TCR repertoire or identify and track more than a few antigen-specific T cells in mice or humans. For mouse studies, this had led to a reliance on model antigens and TCR transgenes. The study of limited human clinical samples, in contrast, requires techniques that can simultaneously survey phenotype, function and reactivity to many T cell epitopes. Thanks to recent advances in single-cell and cytometry methodologies, as well as high-throughput sequencing of the TCR repertoire, we now have or will soon have the tools needed to comprehensively analyze T-cell responses during health and disease.
doi:10.1038/nbt.2783
PMCID: PMC4001742  PMID: 24441473

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