Predictions of epitopes presented by class II human leukocyte antigen molecules (HLA-II) have limited accuracy, restricting vaccine and therapy design. Here we combined unbiased mass spectrometry ...with a motif deconvolution algorithm to profile and analyze a total of 99,265 unique peptides eluted from HLA-II molecules. We then trained an epitope prediction algorithm with these data and improved prediction of pathogen and tumor-associated class II neoepitopes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that ...non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.
Mutation-derived neoantigens are now established as attractive targets for cancer immunotherapy. The field of adoptive T cell transfer (ACT) therapy was significantly reshaped by tumor neoantigens ...and is now moving towards the genetic engineering of T cells with neoantigen-specific T cell receptors (TCRs). Yet, the identification of neoantigen-reactive TCRs remains challenging and the process needs to be adapted to clinical timelines. In addition, the state of recipient T cells for TCR transduction is critical and can affect TCR-ACT efficacy. Here we provide an overview of the main strategies for TCR-engineering, describe the selection and expansion of optimal carrier cells for TCR-ACT and discuss the next-generation methods for rapid identification of relevant TCR candidates for gene transfer therapy.
The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. Here, we characterize the T cell receptor (TCR) functional (antigen sensitivity) and structural ...(monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy donors. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCR biophysicochemical properties, we derive and apply an in silico model predicting TCR structural avidity and validate the enrichment in high avidity T cells in patients' tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy.
Among them, the identification and validation of clinically-relevant neoantigens require a complex, individualized and costly approach which only yields a limited number of neoepitope per patient.4,5 ...We recently reported a novel method that enables the sensitive detection of tumour neoantigens in highly enriched in vitro-expanded tumour-infiltrating lymphocytes (TILs).6 This strategy, dubbed NeoScreen, is based on the early exposure of patients’ tumours to private sets of selected neoantigens loaded on competent autologous B cells. Among the many possible metabolic interventions (Figure 1) are the addition of memory cytokines during T cell expansion,21 the inhibition of signalling pathways leading to T cell effector differentiation22 or the prevention of T cell activation-induced cell death by supplementation with anti-oxidants.23 In addition, TILs can be re-programmed towards memory and stemness by direct genetic engineering.22,24 Importantly, the timing and combinations of manufacturing-compatible interventions, either pharmacological or genetic, remain to be investigated. Of interest, TIL therapy may be combined with standard-of-care cancer treatments such as chemotherapy and radiotherapy which can release tumour antigens, thus recruiting cellular immunity.25 Alternatively, a prime-boost strategy including prior vaccination could first allow the mobilization of tumour-specific T cells and then TIL therapy could synergistically enhance anti-tumour immunity.3,25 Moreover, vaccination may be used as a boost following ACT in order to promote efficient re-stimulation and infiltration of adoptively transferred TILs in vivo.26 Overall, the establishment of relevant combination strategies should foster significant improvement in clinical benefit.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Droplet microfluidics are characterized by the generation and manipulation of discrete volumes of solutions, generated with the use of immiscible phases. Those droplets can then be controlled, ...transported, analyzed or their content modified. In this wide droplet microfluidic toolbox, no means are available to generate, in a controlled manner, droplets co-encapsulating to aqueous phases. Indeed, current methods rely on random co-encapsulation of two aqueous phases during droplet generation or the merging of two random droplets containing different aqueous phases. In this study, we present a novel droplet microfluidic device to reliably and efficiently co-encapsulate two different aqueous phases in micro-droplets. In order to achieve this, we combined existing droplet microfluidic modules in a novel way. The different aqueous phases are individually encapsulated in droplets of different sizes. Those droplet populations are then filtered in order to position each droplet type towards its adequate trapping compartment in traps of a floating trap array. Single droplets, each containing a different aqueous phase, are thus paired and then merged. This pairing at high efficiency is achieved thanks to a unique combination of floating trap arrays, a droplet railing system and a droplet size-based filtering mechanism. The microfluidic chip design presented here provides a filtering threshold with droplets larger than 35 μm (big droplets) being deviated to the lower rail while droplets smaller than 20 μm (small droplets) remain on the upper rail. The effects of the rail height and the distance between the two (upper and lower) rails were investigated. The optimal trap dimensions provide a trapping efficiency of 100% for small and big droplets with a limited double trapping (both compartments of the traps filled with the same droplet type) of 5%. The use of electrocoalescence enables the generation of a droplet while co-encapsulating two aqueous phases. Using the presented microfluidic device libraries of 300 droplets, dual aqueous content can be generated in less than 30 min.
Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the ...mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine.
We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration.
A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current.
A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity.
•A neurochemically diverse group of mouse and human hypothalamic neurons rapidly sense and respond to l-leucine.•Leucine can produce neuronal activation or neuronal inhibition via distinct and novel Ca2+ signaling mechanisms.•Leucine activates 25% ARH POMC neurons.•Leucine inhibits 10% ARH NPY/AGRP neurons and reduces AGRP secretion from fasted mediobasal hypothalamic slices.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Innovative vaccine products will be critical in helping to address the existing implementation barriers that have prevented the achievement of the measles and rubella (MR) vaccine coverage targets. ...Overcoming those barriers will be necessary to achieve the "Immunization Agenda 2030" goals. Microarray patches (MAPs), an innovative needle-free delivery device currently in clinical development, can be a potential game changer in this respect and contribute to the equitable delivery of vaccines in low- and middle-income countries and pandemic preparedness and response. Developing in-depth knowledge of the most desired and impactful uses of MRMAPs can prove critical to identifying the critical attributes of the target product profile, informing policy and adoption decisions, and helping to evaluate the potential public health and economic value of this technology. The first step in this process is the definition of the potential use cases for MR-MAPs, i.e., where and how this product is most likely to be used within the immunization programme.
By applying a design-based user-centric approach, we implemented a three-step process, including a desk review, a survey, and interviews, to define the most relevant use cases for MR MAPS.
Six use cases have been identified as relevant across all different countries and immunization programme designs and validated by experts.
The identified use cases have already informed the demand estimate for MR-MAPs and provided the foundation for developing an initial full vaccine value assessment. We believe that, in the future, they will be highly valuable in ensuring that the roll-out of this promising innovation is designed in a way that maximizes the impact, particularly in populations and countries that are most in need.
Progress toward measles and rubella (MR) elimination has stagnated as countries are unable to reach the required 95% vaccine coverage. Microarray patches (MAPs) are anticipated to offer significant ...programmatic advantages to needle and syringe (N/S) presentation and increase MR vaccination coverage. A demand forecast analysis of the programmatic doses required (PDR) could accelerate MR-MAP development by informing the size and return of the investment required to manufacture MAPs.
Unconstrained global MR-MAP demand for 2030-2040 was estimated for three scenarios, for groups of countries with similar characteristics (archetypes), and four types of uses of MR-MAPs (use cases). The base scenario 1 assumed that MR-MAPs would replace a share of MR doses delivered by N/S, and that MAPs can reach a proportion of previously unimmunised populations. Scenario 2 assumed that MR-MAPs would be piloted in selected countries in each region of the World Health Organization (WHO); and scenario 3 explored introduction of MR-MAPs earlier in countries with the lowest measles vaccine coverage and highest MR disease burden. We conducted sensitivity analyses to measure the impact of data uncertainty.
For the base scenario (1), the estimated global PDR for MR-MAPs was forecasted at 30 million doses in 2030 and increased to 220 million doses by 2040. Compared to scenario 1, scenario 2 resulted in an overall decrease in PDR of 18%, and scenario 3 resulted in a 21% increase in PDR between 2030 and 2040. Sensitivity analyses revealed that assumptions around the anticipated reach or coverage of MR-MAPs, particularly in the hard-to-reach and MOV populations, and the market penetration of MR-MAPs significantly impacted the estimated PDR.
Significant demand is expected for MR-MAPs between 2030 and 2040, however, efforts are required to address remaining data quality, uncertainties and gaps that underpin the assumptions in this analysis.
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