Towards a Swiss health study with human biomonitoring: Learnings from the pilot phase about participation and design.

BACKGROUND: A large-scale national cohort aiming at investigating the health status and determinants in the general population is essential for high-quality public health research and regulatory decision-making. We present the protocol and first results of the pilot phase to a Swiss national cohort aiming at establishing the study procedures, evaluating feasibility, and assessing participation and willingness to participate. METHODS: The pilot phase 2020/21 included 3 components recruited via different channels: a population-based cross-sectional study targeting the adult population (20-69 years) of the Vaud and Bern cantons via personal invitation, a sub-study on selenium in a convenience sample of vegans and vegetarians via non-personal invitation in vegan/vegetarian networks, and a self-selected sample via news promotion (restricted protocol). Along with a participatory approach and participation, we tested the study procedures including online questionnaires, onsite health examination, food intake, physical activity assessments and biosample collection following high-quality standards. RESULTS: The population-based study and the selenium sub-study had 638 (participation rate: 14%) and 109 participants, respectively, both with an over-representation of women. Of altogether 1349 recruited participants over 90% expressed interest in participating to a national health study, over 75% to contribute to medicine progress and help improving others' health, whereas about one third expressed concerns over data protection and data misuse. CONCLUSIONS: Publicly accessible high-quality public health data and human biomonitoring samples were collected. There is high interest of the general population in taking part in a national cohort on health. Challenges reside in achieving a higher participation rate and external validity. For project management clear governance is key.

Blood DNA methylation signatures of lifestyle exposures: tobacco and alcohol consumption.

BACKGROUND: Smoking and alcohol consumption may compromise health by way of epigenetic modifications. Epigenetic signatures of alcohol and tobacco consumption could provide insights into the reversibility of phenotypic changes incurred with differing levels of lifestyle exposures. This study describes and validates two novel epigenetic signatures of tobacco (EpiTob) and alcohol (EpiAlc) consumption and investigates their association with disease outcomes. METHODS: The epigenetic signatures, EpiTob and EpiAlc, were developed using data from the Swiss Kidney Project on Genes in Hypertension (SKIPOGH) (N = 689). Epigenetic and phenotypic data available from the 1921 (N = 550) and 1936 (N = 1091) Lothian Birth Cohort (LBC) studies, and two publicly available datasets on GEO Accession (GSE50660, N = 464; and GSE110043, N = 94) were used to validate the signatures. A multivariable logistic regression model, adjusting for age and sex, was used to assess the association between self-reported tobacco or alcohol consumption and the respective epigenetic signature, as well as to estimate the association between CVD and epigenetic signatures. A Cox proportional hazard model was used to estimate the risk of mortality in association with the EpiTob and EpiAlc signatures. RESULTS: The EpiTob signature was positively associated with self-reported tobacco consumption for current or never smokers with explained variance ranging from 0.49 (LBC1921) to 0.72 (LBC1936) (pseudo-R2). In the SKIPOGH, LBC1921 and LBC1936 cohorts, the epigenetic signature for alcohol consumption explained limited variance in association with self-reported alcohol status [i.e., non-drinker, moderate drinker, and heavy drinker] (pseudo-R2 = 0.05, 0.03 and 0.03, respectively), although this improved considerably when measuring self-reported alcohol consumption with standardized units consumed per week (SKIPOGH R2 = 0.21; LBC1921 R2 = 0.31; LBC1936 R2 = 0.41). Both signatures were associated with history of CVD in SKIPOGH and LBC1936, but not in LBC1921. The EpiTob signature was associated with increased risk of all-cause and lung-cancer specific mortality in the 1936 and 1921 LBC cohorts. CONCLUSIONS: This study found the EpiTob and EpiAlc signatures to be well-correlated with self-reported exposure status and associated with long-term health outcomes. Epigenetic signatures of lifestyle exposures may reduce measurement issues and biases and could aid in risk stratification for informing early-stage targeted interventions.

Overproduction of IL-2 by Cbl-b deficient CD4+ T cells provides resistance against regulatory T cells.

Regulatory T cells are integral to the regulation of autoimmune and anti-tumor immune responses. However, several studies have suggested that changes in T cell signaling networks can result in T cells that are resistant to the suppressive effects of regulatory T cells. Here, we investigated the role of Cbl-b, an E3 ubiquitin ligase, in establishing resistance to Treg-mediated suppression. We found that the absence of Cbl-b, a negative regulator of multiple TCR signaling pathways, rendered T cells impartial to Treg suppression by regulating cytokine networks leading to improved anti-tumor immunity despite the presence of Treg cells in the tumor. Specifically, Cbl-b KO CD4+FoxP3- T cells hyper-produced IL-2 and together with IL-2 Rα upregulation served as an essential mechanism to escape suppression by Treg cells. Furthermore, we report that IL-2 serves as the central molecule required for cytokine-induced Treg resistance. Collectively our data emphasize the role of IL-2 as a key mechanism that renders CD4+ T cells resistant to the inhibitory effects of Treg cells.

NK Cells Regulate CD8+ T Cell Mediated Autoimmunity.

Elucidating key factors that regulate immune-mediated pathology in vivo is critical for developing improved strategies to treat autoimmune disease and cancer. NK cells can exhibit regulatory functions against CD8+ T cells following viral infection. Here we show that while low doses of lymphocytic choriomeningitis virus (LCMV-WE) can readily induce strong CD8+ T cell responses and diabetes in mice expressing the LCMV glycoprotein on ß-islet cells (RIP-GP mice), hyperglycemia does not occur after infection with higher doses of LCMV. High-dose LCMV infection induced an impaired CD8+ T cell response, which coincided with increased NK cell activity during early time points following infection. Notably, we observed increased NKp46 expression on NK cells during infection with higher doses, which resulted in an NK cell dependent suppression of T cells. Accordingly, depletion with antibodies specific for NK1.1 as well as NKp46 deficiency (Ncr1gfp/gfp mice) could restore CD8+ T cell immunity and permitted the induction of diabetes even following infection of RIP-GP mice with high-dose LCMV. Therefore, we identify conditions where innate lymphoid cells can play a regulatory role and interfere with CD8+ T cell mediated tissue specific pathology using an NKp46 dependent mechanism.

High participation rate among 25 721 patients with broad age range in a hospital-based research project involving whole-genome sequencing - the Lausanne Institutional Biobank.

AIMS: We aimed to evaluate the interest of adult inpatients and selected outpatients in engaging in a large, real-life, hospital-based, genomic medicine research project and in receiving clinically actionable incidental findings. METHODS: Within the framework of the cross-sectional Institutional Biobank of Lausanne, Switzerland, a total of 25721 patients of the CHUV University Hospital were systematically invited to grant researchers access to their biomedical data and to donate blood for future analyses, including whole-genome sequencing. Multivariable logistic regression analysis was used to identify personal factors, including age, gender, religion, ethnicity, citizenship, education level and mode of admission, associated with willingness to participate in this genomic research project and with interest in receiving clinically actionable incidental findings. RESULTS: The overall participation rate was 79% (20343/25721). Participation rate declined progressively with age, averaging 83%, 75%, 67% and 62% in patients aged <64 years (n = 13108), ≥64 years (n = 12613), ≥80 years (n = 4557) and ≥90 years (n = 1050), respectively. Factors associated with participation substantially differed between age strata. Patients less likely to participate included women (odds ratio 0.86, [95% confidence interval 0.79-0.95] and 0.78 [0.71-0.85] before and after age 64, respectively), non-Swiss (0.81 [0.74-0.90] and 0.58 [0.52-0.65]) and those admitted through the emergency ward (0.88 [0.79-0.98] and 0.66 [0.60-0.73]). Religion and marital status were associated with participation among patients aged <64 years. A total of 19 018 (93%) participants were willing to be re-contacted for incidental findings. A high education level was associated with higher participation rate, but not with higher willingness to receive incidental findings within the population who had agreed to participate. CONCLUSION: A large proportion of adult patients, even among the elderly, are willing to actively participate and receive incidental findings in this systematic hospital-based precision and genomic medicine research program with broad consent.

Identification and molecular characterisation of Lausanne Institutional Biobank participants with familial hypercholesterolaemia - a proof-of-concept study.

AIMS: We aimed to identify familial hypercholesterolaemia mutation carriers among participants to the Lausanne Institutional Biobank (BIL). Our experimental workflow was designed as a proof-of-concept demonstration of the resources and services provided by our integrated institutional clinical research support platform. METHODS: Familial hypercholesterolaemia was used as a model of a relatively common yet often underdiagnosed and inadequately treated Mendelian disease. Clinical and laboratory information was extracted from electronic hospital records. Patients were selected using elevated plasma cholesterol levels (total cholesterol ≥7.5 mM or low-density lipoprotein cholesterol ≥5 mM), premature coronary artery disease status and age (18-60 yr) as main inclusion criteria. LDLR, APOB and PCSK9 were analysed by high-throughput DNA sequencing. The most relevant mutations were confirmed by Sanger sequencing. RESULTS: Of 23 737 patients contacted by the BIL, 17 760 individuals consented to participate and 13 094 wished to be recontacted if there were findings requiring clinical action. Plasma cholesterol records were available for 5111 participants, of whom 94 were selected for genetic screening. Twenty-five of the tested patients presented with premature coronary artery disease while 69 had no such diagnosis. Seven heterozygous carriers of eight rare coding missense variants were identified. Three mutations were pathogenic (APOB p.R3527Q) or likely pathogenic (LDLR p.C27W, LDLR p.P526S) for hypercholesterolaemia, while the others were either benign or of unknown significance. One patient was a double heterozygote for variants APOB p.R3527Q and LDLR p.P526S. CONCLUSION: This work illustrates how clinical and translational research can benefit from a dedicated platform integrating both a hospital-based biobank and a data support team.

c-Rel phenocopies PKCtheta but not Bcl-10 in regulating CD8+ T-cell activation versus tolerance.

Elucidating the signaling events that promote T-cell tolerance versus activation provides important insights for manipulating immunity in vivo. Previous studies have suggested that the absence of PKCtheta results in the induction of anergy and that the balance between the induction of the transcription factors NFAT, AP1 and NF-kappaB plays a key role in determining whether T-cell anergy or activation is induced. Here, we examine whether Bcl-10 and specific family members of NF-kappaB act downstream of PKCtheta to alter CD8(+) T-cell activation and/or anergy. We showed that T cells from mice deficient in c-Rel but not NF-kappaB1 (p50) have increased susceptibility to the induction of anergy, similar to T cells from PKCtheta-deficient mice. Surprisingly T cells from Bcl-10-deficient mice showed a strikingly different phenotype to the PKCtheta-deficient T cells, with a severe block in TCR-mediated activation. Furthermore, we have also shown that survival signals downstream of NF-kappaB, are uncoupled from signals that mediate T-cell anergy. These results suggest that c-Rel plays a critical role downstream of PKCtheta in controlling CD8(+) T-cell anergy induction.

IL-7 adjuvant treatment enhances long-term tumor-antigen-specific CD8+ T-cell responses after immunization with recombinant lentivector.

Immunization with recombinant lentivector elicits higher frequencies of tumor antigen-specific memory CD8+ T cells than peptide-based vaccines. This finding correlates with our observation that, upon recombinant lentivector immunization, a higher fraction of antigen-specific effector CD8+ T cells does not down-regulate the expression of the survival/memory marker interleukin-7 receptor alpha chain (IL-7Ralpha). Here we show that, surprisingly, higher expression of IL-7Ralpha on recombinant lentivector-induced effector CD8+ T cells does not result in the up-regulation of survival molecules, such as Bcl-2. We thus hypothesized that physiologic levels of IL-7 might be limiting in vivo for delivering survival signals to the expanding population of effector cells. To test this hypothesis, we administered recombinant IL-7 during the effector phase of the response. We observed an up-regulation of Bcl-2 and a strong expansion of antigen-specific effector CD8+ T cells, and of naive CD8+ T cells. Strikingly, IL-7 treatment elicited also a significant increase in the number of antigen-specific memory CD8+ T cells in recombinant lentivector-immunized mice, but not in peptide-immunized mice. Altogether, these data show that IL-7 adjuvant treatment can enhance long-term antigen-specific CD8+ T-cell responses. However, its efficacy depends on the expression of IL-7Ralpha at the surface of effector CD8+ T cells.

Lentivector immunization induces tumor antigen-specific B and T cell responses in vivo.

Expression of the cancer/germ-line antigen NY-ESO-1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY-ESO-1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA-A2-transgenic mice elicited long-lasting B and T cell responses against NY-ESO-1. CD8+ T cells against the HLA-A2-restricted peptide NY-ESO-1(157-165) were readily detectable ex vivo and showed restricted TCR Vbeta usage. Moreover, rLV/ESO elicited a far greater anti-NY-ESO-1(157-165) CD8+ T cell response than peptide- or protein-based vaccines. Anti-NY-ESO-1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen-specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY-ESO-1. The induced CD4+ T cells were primarily directed against a single NY-ESO-1 epitope spanning amino acids 81-100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.

Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses.

Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.

Impact of orthologous melan-A peptide immunizations on the anti-self melan-A/HLA-A2 T cell cross-reactivity.

In HLA-A2 individuals, the CD8 T cell response against the differentiation Ag Melan-A is mainly directed toward the peptide Melan-A26-35. The murine Melan-A24-33 sequence encodes a peptide that is identical with the human Melan-A26-35 decamer, except for a Thr-to-Ile substitution at the penultimate position. Here, we show that the murine Melan-A24-33 is naturally processed and presented by HLA-A2 molecules. Based on these findings, we compared the CD8 T cell response to human and murine Melan-A peptide by immunizing HLA-A2 transgenic mice. Even though the magnitude of the CTL response elicited by the murine Melan-A peptide was lower than the one elicited by the human Melan-A peptide, both populations of CTL recognized the corresponding immunizing peptide with the same functional avidity. Interestingly, CTL specific for the murine Melan-A peptide were completely cross-reactive against the orthologous human peptide, whereas anti-human Melan-A CTL recognized the murine Melan-A peptide with lower avidity. Structurally, this discrepancy could be explained by the fact that Ile32 of murine Melan-A24-33 created a larger TCR contact area than Thr34 of human Melan-A26-35. These data indicate that, even if immunizations with orthologous peptides can induce strong specific T cell responses, the quality of this response against syngeneic targets might be suboptimal due to the structure of the peptide-TCR contact surface.

Efficient induction of tumor antigen-specific CD8+ memory T cells by recombinant lentivectors.

The success of active cancer immunotherapy entails a robust induction of tumor-reactive effector and memory CD8+ T cells. We compared the in vivo immunogenicity of the melanoma-associated antigen Melan-A(26-35) encoded by third-generation recombinant lentivector (rec. lv) or as peptide admixed with a strong adjuvant. Ex vivo analyses of immunized HLA-A2/H-2K(b) mice showed that rec. lv triggered a stronger anti-Melan-A CD8+ T -cell response than peptide vaccine. Importantly, the majority of anti-Melan-A T cells elicited by rec. lv expressed the memory marker CD127 at the peak of the primary response. In those mice, memory T cells were detectable several months after priming and could be activated by recall peptide vaccination. These results show that immunization with rec. lv induces not only a strong antigen-specific CD8+ T -cell response but also a long-lasting T-cell memory against a bona fide tumor-associated antigen.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells.

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells.

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.

In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8(+) T cell responses.

The present study evaluates the potential of third-generation lentivirus vectors with respect to their use as in vivo-administered T cell vaccines. We demonstrate that lentivector injection into the footpad of mice transduces DCs that appear in the draining lymph node and in the spleen. In addition, a lentivector vaccine bearing a T cell antigen induced very strong systemic antigen-specific cytotoxic T lymphocyte (CTL) responses in mice. Comparative vaccination performed in two different antigen models demonstrated that in vivo administration of lentivector was superior to transfer of transduced DCs or peptide/adjuvant vaccination in terms of both amplitude and longevity of the CTL response. Our data suggest that a decisive factor for efficient T cell priming by lentivector might be the targeting of DCs in situ and their subsequent migration to secondary lymphoid organs. The combination of performance, ease of application, and absence of pre-existing immunity in humans make lentivector-based vaccines an attractive candidate for cancer immunotherapy.

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors.

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.