Natural variation of ncHLAII molecules: challenges and perspectives

Identifying causal genetic variants for a given phenotype improves diagnosis and provides insights for designing therapies. However, defining the link and/or contribution to a phenotype of rare variants or those with an indirect contribution to disease remains challenging. These variants, however, are key for personalized and precision medicine strategies [1]. In this context, human leukocyte antigens of class II alleles (HLAIIs) facilitate activation of CD4+ T cells and are directly linked to various phenotypes. Non-classical HLAII (ncHLAII) molecules modulate HLAII function and their impact on T-cell responses. The contribution of ncHLAII to phenotypes has been investigated in knockout vs. wild-type cellular and animal models, and recent studies have shown that genetic variants altering the protein-coding regions of these molecules contribute to tuning of HLAII function [2]. These findings suggest that ncHLAII natural variants may play a key role in modulating immune mechanisms that affect human phenotypes

Peptide editing and its modulation

The nonclassical major histocompatibility complex of class II molecules (ncMHCII) HLA‐DM (DM) and HLA‐DO (DO) feature essential functions for the selection of the peptides that are displayed by classical MHCII proteins (MHCII) for CD4+ Th cell surveillance. Thus, although the binding groove of classical MHCII dictates the main features of the peptides displayed, ncMHCII function defines the preferential loading of peptides from specific cellular compartments and the extent to which they are presented. DM acts as a chaperone for classical MHCII molecules facilitating peptide exchange and thereby favoring the binding of peptide‐MHCII complexes of high kinetic stability mostly in late endosomal compartments. DO on the other hand binds to DM blocking its peptide‐editing function in B cells and thymic epithelial cells, limiting DM activity in these cellular subsets. DM and DO distinct expression patterns therefore define specific antigen presentation profiles that select unique peptide pools for each set of antigen presenting cell. We have come a long way understanding the mechanistic underpinnings of such distinct editing profiles and start to grasp the implications for ncMHCII biological function. DM acts as filter for the selection of immunodominant, pathogen‐derived epitopes while DO blocks DM activity under certain physiological conditions to promote tolerance to self. Interestingly, recent findings have shown that the unexplored and neglected ncMHCII genetic diversity modulates retroviral infection in mouse, and affects human ncMHCII function. This review aims at highlighting the importance of ncMHCII function for CD4+ Th cell responses while integrating and evaluating what could be the impact of distinct editing profiles because of natural genetic variations

Genetic Association Besides Classical HLA Class II Genes

Type 1 diabetes is an autoimmune disease with rising incidence in high-income countries. Genetic and environmental predisposing factors contribute to the etiology of the disease, although their interaction is not sufficiently understood to allow for preventive action. Strongest known associations with genetic variation map to classical HLA class II genes. Because of its genetic complexity, the HLA region has been under-represented in genome-wide association studies, having potentially hindered the identification of relevant associations underlying the etiology of the disease. Here, we performed a comprehensive HLA-wide genetic association analysis of type 1 diabetes including multi-allelic and rare variants. We used high-density whole-exome sequencing data of the HLA region in the large UK Biobank dataset to apply gene-based association tests with a carefully defined type 1 diabetes phenotype (97 cases and 48,700 controls). Exon-based and single-variant association tests were used to complement the analysis. We replicated the known association of type 1 diabetes with the classical HLA-DQ gene. Tailoring the analysis toward rare variants, we additionally identified the lysine methyl transferase EHMT2 as associated. Deeper insight into genetic variation associated with disease as presented and discussed in detail here can help unraveling mechanistic details of the etiology of type 1 diabetes. More specifically, we hypothesize that genetic variation in EHMT2 could impact autoimmunity in type 1 diabetes development

CD4+ Th immunogenicity of the Ascaris spp. secreted products

Ascaris spp. is a major health problem of humans and animals alike, and understanding the immunogenicity of its antigens is required for developing urgently needed vaccines. The parasite-secreted products represent the most relevant, yet complex (>250 proteins) antigens of Ascaris spp. as defining the pathogen-host interplay. We applied an in vitro antigen processing system coupled to quantitative proteomics to identify potential CD4+ Th cell epitopes in Ascaris-secreted products. This approach considerably restricts the theoretical list of epitopes using conventional CD4+ Th cell epitope prediction tools. We demonstrate the specificity and utility of our approach on two sets of candidate lists, allowing us identifying hits excluded by either one or both computational methods. More importantly, one of the candidates identified experimentally, clearly demonstrates the presence of pathogen-reactive T cells in healthy human individuals against these antigens. Thus, our work pipeline identifies the first human T cell epitope against Ascaris spp. and represents an easily adaptable platform for characterization of complex antigens, in particular for those pathogens that are not easily amenable for in vivo experimental validation

Sortase-Mediated Multi-Fragment Assemblies by Ligation Site Switching

Sortase-mediated ligation (SML) is a powerful tool of protein chemistry allowing the ligation of peptides containing LPxTG sorting motifs and N-terminal glycine nucleophiles. The installation of a sorting motif into the product prohibits the assembly of multiple fragments by SML. Here we report multi-fragment SML based on switchable sortase substrates. Substitution of the Leu residue by disulfide-containing Cys(StBu) results in active sorting motifs, which are inactivatable by reduction. In combination with a photo-protected N-Gly nucleophile, multi-fragment SML is enabled by repetitive cycles of SML and ligation site switching. The feasibility of this approach was demonstrated by a proof-of-concept four-fragment ligation, the assembly of peptide probes for bivalent chromatin binding proteins and oligomerization of peptide antigens. Biochemical and immuno-assays demonstrated functionality of these probes rendering them promising tools for immunology and chromatin biochemistry

Dynamic palmitoylation events following T-cell receptor signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide–MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease

Extensión del método de modos acoplados para el estudio de medios quirales ilimitados

The Coupled–Mode Method (CMM) is a semi–numerical method in the frequency domain for studying electromagnetic propagation, originally formulated for closed structures, widely used in the literature. In this paper, the formulation of the CMM is extended to the particular case of wave propagation in an unbounded isotropic chiral media (ICM) and the propagation constants and the electromagnetic field components are obtained numerically. To test the accuracy of the approximated results, comparisons with the analytical results are established

Cellular and humoral functional responses after BNT162b2 mRNA vaccination differ longitudinally between naive and subjects recovered from COVID-19

We have analyzed BNT162b2 vaccine-induced immune responses in naive subjects and individuals recovered from coronavirus disease 2019 (COVID-19), both soon after (14 days) and later after (almost 8 months) vaccination. Plasma spike (S)-specific immunoglobulins peak after one vaccine shot in individuals recovered from COVID-19, while a second dose is needed in naive subjects, although the latter group shows reduced levels all along the analyzed period. Despite how the neutralization capacity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mirrors this behavior early after vaccination, both groups show comparable neutralizing antibodies and S-specific B cell levels late post-vaccination. When studying cellular responses, naive individuals exhibit higher SARS-CoV-2-specific cytokine production, CD4+ T cell activation, and proliferation than do individuals recovered from COVID-19, with patent inverse correlations between humoral and cellular variables early post-vaccination. However, almost 8 months post-vaccination, SARS-CoV-2-specific responses are comparable between both groups. Our data indicate that a previous history of COVID-19 differentially determines the functional T and B cell-mediated responses to BNT162b2 vaccination over time.C.d.F., J.G.-P., and J.A. are supported by Instituto de Salud Carlos III (ISCII). We thank JM Ligos and Cytek Biosciences for their technical support. Research in E.L.-C.’s lab was supported by Fundación Familia Alonso, Santander Bank, Real Seguros, Fundación Mutua Madrileña, Fundación Uria, Fundación La Caixa, and Ayuntamiento de Madrid.S

Análisis teórico–experimental de medios quirales isótropos en guía rectangular

In this communication a description of the hybrid technique combining the coupled mode method (CMM) and the mode matching method (MMM) is presented. This numerical tool is suitable for the analysis of general 3D structures implemented in rectangular waveguide. Also, this formulation is independent of the number of discontinuities of the structure. As an example, the analysis of a 3D structure formed by the arrangement of a waveguide section filled with an isotropic chiral material and inserted between to empty waveguide sections is proposed. Finally, the structure has been implemented in a rectangular waveguide and comparisons between the theoretic and experimental results are established