Shaping of infant B cell receptor repertoires by environmental factors and infectious disease.
Science translational medicine
2019; 11 (481)
Antigenic exposures at epithelial sites in infancy and early childhood are thought to influence the maturation of humoral immunity and modulate the risk of developing immunoglobulin E (IgE)-mediated allergic disease. How different kinds of environmental exposures influence B cell isotype switching to IgE, IgG, or IgA, and the somatic mutation maturation of these antibody pools, is not fully understood. We sequenced antibody repertoires in longitudinal blood samples in a birth cohort from infancy through the first 3 years of life and found that, whereas IgG and IgA show linear increases in mutational maturation with age, IgM and IgD mutations are more closely tied to pathogen exposure. IgE mutation frequencies are primarily increased in children with impaired skin barrier conditions such as eczema, suggesting that IgE affinity maturation could provide a mechanistic link between epithelial barrier failure and allergy development.
View details for PubMedID 30814336
Increased T Cell Differentiation and Cytolytic Function in Bangladeshi Compared to American Children.
Frontiers in immunology
2019; 10: 2239
During the first 5 years of life, children are especially vulnerable to infection-related morbidity and mortality. Conversely, the Hygiene Hypothesis suggests that a lack of exposure to infectious agents early in life could explain the increasing incidence of allergies and autoimmunity in high-income countries. Understanding these phenomena, however, is hampered by a lack of comprehensive, direct immune monitoring in children with differing degrees of microbial exposure. Using mass cytometry, we provide an in-depth profile of the peripheral blood mononuclear cells (PBMCs) of children in regions at the extremes of exposure: the San Francisco Bay Area, USA and an economically poor district of Dhaka, Bangladesh. Despite variability in clinical health, functional characteristics of PBMCs were similar in Bangladeshi and American children at 1 year of age. However, by 2-3 years of age, Bangladeshi children's immune cells often demonstrated altered activation and cytokine production profiles upon stimulation with PMA-ionomycin, with an overall immune trajectory more in line with American adults. Conversely, immune responses in children from the US remained steady. Using principal component analysis, donor location, ethnic background, and cytomegalovirus infection status were found to account for some of the variation identified among samples. Within Bangladeshi 1-year-olds, stunting (as measured by height-for-age z-scores) was found to be associated with IL-8 and TGFβ expression in PMA-ionomycin stimulated samples. Combined, these findings provide important insights into the immune systems of children in high vs. low microbial exposure environments and suggest an important role for IL-8 and TGFβ in mitigating the microbial challenges faced by the Bangladeshi children.
View details for DOI 10.3389/fimmu.2019.02239
View details for PubMedID 31620139
View details for PubMedCentralID PMC6763580
Live Cell Barcoding for Efficient Analysis of Small Samples by Mass Cytometry.
Methods in molecular biology (Clifton, N.J.)
2019; 1989: 125–35
In mass cytometry, sample loss is of considerable concern due to the relative inefficiency of cell event collection compared to similar techniques such as flow cytometry. Cell stimulation and the harsh conditions required in the later stages of certain sample preparations also contribute to cell loss. Low starting cell numbers are especially susceptible to these effects, potentially limiting the ability to use mass cytometry. Here is presented a live cell barcoding scheme and additional efficiency methods to improve recovery and achieve consistent staining for small samples.
View details for PubMedID 31077103
Acute myeloid leukemia immunopeptidome reveals HLA presentation of mutated nucleophosmin.
2019; 14 (7): e0219547
Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapeutic approaches. Here we sought to study the HLA Class I and II immunopeptidome of thirteen primary AML tumor samples and two AML cell lines (OCI-AML3 and MV4-11) using mass spectrometry to evaluate for endogenous mutation-bearing HLA ligands from common shared AML mutations. We identified two endogenous, mutation-bearing HLA Class I ligands from nucleophosmin (NPM1). The ligands, AVEEVSLRK from two patient samples and C(cys)LAVEEVSL from OCI-AML3, are predicted to bind the common HLA haplotypes, HLA-A*03:01 and HLA-A*02:01 respectively. Since NPM1 is mutated in approximately one-third of patients with AML, the finding of endogenous HLA ligands from mutated NPM1 supports future studies evaluating immunotherapeutic approaches against this shared target, for this subset of patients with AML.
View details for DOI 10.1371/journal.pone.0219547
View details for PubMedID 31291378
Predicting HLA class II antigen presentation through integrated deep learning.
Accurate prediction of antigen presentation by human leukocyte antigen (HLA) class II molecules would be valuable for vaccine development and cancer immunotherapies. Current computational methods trained on in vitro binding data are limited by insufficient training data and algorithmic constraints. Here we describe MARIA (major histocompatibility complex analysis with recurrent integrated architecture; https://maria.stanford.edu/ ), a multimodal recurrent neural network for predicting the likelihood of antigen presentation from a gene of interest in the context of specific HLA class II alleles. In addition to in vitro binding measurements, MARIA is trained on peptide HLA ligand sequences identified by mass spectrometry, expression levels of antigen genes and protease cleavage signatures. Because it leverages these diverse training data and our improved machine learning framework, MARIA (area under the curve = 0.89-0.92) outperformed existing methods in validation datasets. Across independent cancer neoantigen studies, peptides with high MARIA scores are more likely to elicit strong CD4+ T cell responses. MARIA allows identification of immunogenic epitopes in diverse cancers and autoimmune disease.
View details for DOI 10.1038/s41587-019-0280-2
View details for PubMedID 31611695
Histone H3 lysine 4 methylation signature associated with human undernutrition.
Proceedings of the National Academy of Sciences of the United States of America
Chronically undernourished children become stunted during their first 2 years and thereafter bear burdens of ill health for the rest of their lives. Contributors to stunting include poor nutrition and exposure to pathogens, and parental history may also play a role. However, the epigenetic impact of a poor environment on young children is largely unknown. Here we show the unfolding pattern of histone H3 lysine 4 trimethylation (H3K4me3) in children and mothers living in an urban slum in Dhaka, Bangladesh. A pattern of chromatin modification in blood cells of stunted children emerges over time and involves a global decrease in methylation at canonical locations near gene start sites and increased methylation at ectopic sites throughout the genome. This redistribution occurs at metabolic and immune genes and was specific for H3K4me3, as it was not observed for histone H3 lysine 27 acetylation in the same samples. Methylation changes in stunting globally resemble changes that occur in vitro in response to altered methylation capacity, suggesting that reduced levels of one-carbon nutrients in the diet play a key role in stunting in this population. A network of differentially expressed genes in stunted children reveals effects on chromatin modification machinery, including turnover of H3K4me3, as well as posttranscriptional gene regulation affecting immune response pathways and lipid metabolism. Consistent with these changes, reduced expression of the endocytic receptor gene LDL receptor 1 (LRP1) is a driver of stunting in a mouse model, suggesting a target for intervention.
View details for PubMedID 30420518
Advanced model systems and tools for basic and translational human immunology.
2018; 10 (1): 73
There are fundamental differences between humans and the animals we typically use to study the immune system. We have learned much from genetically manipulated and inbred animal models, but instances in which these findings have been successfully translated to human immunity have been rare. Embracing the genetic and environmental diversity of humans can tell us about the fundamental biology of immune cell types and the elasticity of the immune system. Although people are much more immunologically diverse than conventionally housed animal models, tools and technologies are now available that permit high-throughput analysis of human samples, including both blood and tissues, which will give us deep insights into human immunity in health and disease. As we gain a more detailed picture of the human immune system, we can build more sophisticated models to better reflect this complexity, both enabling the discovery of new immunological mechanisms and facilitating translation into the clinic.
View details for PubMedID 30266097
Comparison of CyTOF assays across sites: Results of a six-center pilot study
JOURNAL OF IMMUNOLOGICAL METHODS
2018; 453: 37–43
For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values<30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.
View details for PubMedID 29174717
View details for PubMedCentralID PMC5805584
Effect of IL-7 Therapy on Phospho-Ribosomal Protein S6 and TRAF1 Expression in HIV-Specific CD8 T Cells in Patients Receiving Antiretroviral Therapy
JOURNAL OF IMMUNOLOGY
2018; 200 (2): 558–64
IL-7 therapy has been evaluated in patients who do not regain normal CD4 T cell counts after virologically successful antiretroviral therapy. IL-7 increases total circulating CD4 and CD8 T cell counts; however, its effect on HIV-specific CD8 T cells has not been fully examined. TRAF1, a prosurvival signaling adaptor required for 4-1BB-mediated costimulation, is lost from chronically stimulated virus-specific CD8 T cells with progression of HIV infection in humans and during chronic lymphocytic choriomeningitis infection in mice. Previous results showed that IL-7 can restore TRAF1 expression in virus-specific CD8 T cells in mice, rendering them sensitive to anti-4-1BB agonist therapy. In this article, we show that IL-7 therapy in humans increases the number of circulating HIV-specific CD8 T cells. For a subset of patients, we also observed an increased frequency of TRAF1+ HIV-specific CD8 T cells 10 wk after completion of IL-7 treatment. IL-7 treatment increased levels of phospho-ribosomal protein S6 in HIV-specific CD8 T cells, suggesting increased activation of the metabolic checkpoint kinase mTORC1. Thus, IL-7 therapy in antiretroviral therapy-treated patients induces sustained changes in the number and phenotype of HIV-specific T cells.
View details for DOI 10.4049/jimmunol.1601254
View details for Web of Science ID 000419443900021
View details for PubMedID 29222166
View details for PubMedCentralID PMC5760332
Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens
2017; 543 (7647): 723-?
Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4(+) T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
View details for DOI 10.1038/nature21433
View details for PubMedID 28329770
Intrinsic 4-1BB signals are indispensable for the establishment of an influenza-specific tissue-resident memory CD8 T-cell population in the lung.
The induction of long-lived heterotypic T-cell protection against influenza virus remains elusive, despite the conservation of T-cell epitopes. T-cell protection against influenza is critically dependent on lung-resident memory T cells (Trm). Here we show that intranasal administration of 4-1BBL along with influenza nucleoprotein in a replication-defective adenovirus vector to influenza pre-immune mice induces a remarkably stable circulating effector memory CD8 T-cell population characterized by higher IL-7Rα expression than control-boosted T cells, as well as a substantial lung parenchymal CD69(+) CD8 Trm population, including both CD103(+) and CD103(-) cells. These T-cell responses persist to greater than 200 days post-boost and protect against lethal influenza challenge in aged (year old) mice. The expansion of the nucleoprotein-specific CD8 Trm population during boosting involves recruitment of circulating antigen-specific cells and is critically dependent on local rather than systemic administration of 4-1BBL as well as on 4-1BB on the CD8 T cells. Moreover, during primary influenza infection of mixed bone marrow chimeras, 4-1BB-deficient T cells fail to contribute to the lung-resident Trm population. These findings establish both endogenous and supraphysiological 4-1BBL as a critical regulator of lung-resident memory CD8 T cells during influenza infection.
View details for DOI 10.1038/mi.2016.124
View details for PubMedID 28051085
Identifying specificity groups in the T cell receptor repertoire.
T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.
View details for PubMedID 28636589
High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization.
Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.
View details for PubMedID 29221729
Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals.
2016; 12 (10)
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.
View details for DOI 10.1371/journal.ppat.1005892
View details for PubMedID 27764254
View details for PubMedCentralID PMC5072604
The Human Immune System Recognizes Neopeptides Derived from Mitochondrial DNA Deletions
JOURNAL OF IMMUNOLOGY
2014; 192 (10): 4581-4591
Mutations in mitochondrial (mt) DNA accumulate with age and can result in the generation of neopeptides. Immune surveillance of such neopeptides may allow suboptimal mitochondria to be eliminated, thereby avoiding mt-related diseases, but may also contribute to autoimmunity in susceptible individuals. To date, the direct recognition of neo-mtpeptides by the adaptive immune system has not been demonstrated. In this study we used bioinformatics approaches to predict MHC binding of neopeptides identified from known deletions in mtDNA. Six such peptides were confirmed experimentally to bind to HLA-A*02. Pre-existing human CD4(+) and CD8(+) T cells from healthy donors were shown to recognize and respond to these neopeptides. One remarkably promiscuous immunodominant peptide (P9) could be presented by diverse MHC molecules to CD4(+) and/or CD8(+) T cells from 75% of the healthy donors tested. The common soil microbe, Bacillus pumilus, encodes a 9-mer that differs by one amino acid from P9. Similarly, the ATP synthase F0 subunit 6 from normal human mitochondria encodes a 9-mer with a single amino acid difference from P9 with 89% homology to P9. T cells expanded from human PBMCs using the B. pumilus or self-mt peptide bound to P9/HLA-A2 tetramers, arguing for cross-reactivity between T cells with specificity for self and foreign homologs of the altered mt peptide. These findings provide proof of principal that the immune system can recognize peptides arising from spontaneous somatic mutations and that such responses might be primed by foreign peptides and/or be cross-reactive with self.
View details for DOI 10.4049/jimmunol.1300774
View details for Web of Science ID 000335973600015
View details for PubMedID 24733843
Humoral and Cell-Mediated Immunity to Pandemic H1N1 Influenza in a Canadian Cohort One Year Post-Pandemic: Implications for Vaccination
2011; 6 (11)
We evaluated a cohort of Canadian donors for T cell and antibody responses against influenza A/California/7/2009 (pH1N1) at 8-10 months after the 2nd pandemic wave by flow cytometry and microneutralization assays. Memory CD8 T cell responses to pH1N1 were detectable in 58% (61/105) of donors. These responses were largely due to cross-reactive CD8 T cell epitopes as, for those donors tested, similar recall responses were obtained to A/California 2009 and A/PR8 1934 H1N1 Hviruses. Longitudinal analysis of a single infected individual showed only a small and transient increase in neutralizing antibody levels, but a robust CD8 T cell response that rose rapidly post symptom onset, peaking at 3 weeks, followed by a gradual decline to the baseline levels seen in a seroprevalence cohort post-pandemic. The magnitude of the influenza-specific CD8 T cell memory response at one year post-pandemic was similar in cases and controls as well as in vaccinated and unvaccinated donors, suggesting that any T cell boosting from infection was transient. Pandemic H1-specific antibodies were only detectable in approximately half of vaccinated donors. However, those who were vaccinated within a few months following infection had the highest persisting antibody titers, suggesting that vaccination shortly after influenza infection can boost or sustain antibody levels. For the most part the circulating influenza-specific T cell and serum antibody levels in the population at one year post-pandemic were not different between cases and controls, suggesting that natural infection does not lead to higher long term T cell and antibody responses in donors with pre-existing immunity to influenza. However, based on the responses of one longitudinal donor, it is possible for a small population of pre-existing cross-reactive memory CD8 T cells to expand rapidly following infection and this response may aid in viral clearance and contribute to a lessening of disease severity.
View details for DOI 10.1371/journal.pone.0028063
View details for Web of Science ID 000298162000067
View details for PubMedID 22132212
View details for PubMedCentralID PMC3223223
Influenza-Specific T Cells from Older People Are Enriched in the Late Effector Subset and Their Presence Inversely Correlates with Vaccine Response
2011; 6 (8)
T cells specific for persistent pathogens accumulate with age and express markers of immune senescence. In contrast, much less is known about the state of T cell memory for acutely infecting pathogens. Here we examined T cell responses to influenza in human peripheral blood mononuclear cells from older (>64) and younger (<40) donors using whole virus restimulation with influenza A (A/PR8/34) ex vivo. Although most donors had pre-existing influenza reactive T cells as measured by IFNγ production, older donors had smaller populations of influenza-responsive T cells than young controls and had lost a significant proportion of their CD45RA-negative functional memory population. Despite this apparent dysfunction in a proportion of the older T cells, both old and young donors' T cells from 2008 could respond to A/California/07/2009 ex vivo. For HLA-A2+ donors, MHC tetramer staining showed that a higher proportion of influenza-specific memory CD8 T cells from the 65+ group co-express the markers killer cell lectin-like receptor G1 (KLRG1) and CD57 compared to their younger counterparts. These markers have previously been associated with a late differentiation state or immune senescence. Thus, memory CD8 T cells to an acutely infecting pathogen show signs of advanced differentiation and functional deterioration with age. There was a significant negative correlation between the frequency of KLRG1(+)CD57(+) influenza M1-specific CD8 T cells pre-vaccination and the ability to make antibodies in response to vaccination with seasonal trivalent inactivated vaccine, whereas no such trend was observed when the total CD8(+)KLRG1(+)CD57(+) population was analyzed. These results suggest that the state of the influenza-specific memory CD8 T cells may be a predictive indicator of a vaccine responsive healthy immune system in old age.
View details for DOI 10.1371/journal.pone.0023698
View details for Web of Science ID 000294251800021
View details for PubMedID 21887299
View details for PubMedCentralID PMC3161762
- Harnessing functional food strategies for the health challenges of space travel-Fermented soy for astronaut nutrition ACTA ASTRONAUTICA 2011; 68 (7-8): 731-738
Immunomodulatory Properties of Fermented Soy and Dairy Milks Prepared with Lactic Acid Bacteria
JOURNAL OF FOOD SCIENCE
2009; 74 (8): M423-M430
Fermented soy and dairy milk preparations provide a means for delivering lactic acid bacteria and their fermentation products into the diet. Our aims were to test immunomodulatory bioactivity of fermented soy beverage (SB) and dairy milk blend (MB) preparations on human intestinal epithelial cells (IEC) and to determine the impact of freezing medium on culture survival prior to bioactivity analyses. Fermented SB and MB were prepared using pure or mixed cultures of Streptococcus thermophilus ST5, Bifidobacterium longum R0175, and Lactobacillus helveticus R0052. Immunomodulatory bioactivity was assessed by testing selected SB and MB ferments on tumor necrosis factor alpha (TNFalpha)-treated IEC and measuring effects on Interleukin-8 (IL-8) production. Impact of timing of ferment administration relative to this pro-inflammatory challenge was investigated. The most pronounced reductions in IEC IL-8 production were observed when IEC were treated with either SB or MB ferment preparations prior to TNFalpha challenge. These results indicate that freezing-stable MB and SB ferments prepared with selected strains can modulate IEC IL-8 production in vitro, and suggest that yogurt-like fermented soy formulations could provide a functional food alternative to milk-based fermented products.
View details for DOI 10.1111/j.1750-3841.2009.01308.x
View details for Web of Science ID 000270596100016
View details for PubMedID 19799669