T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.

How T cell receptors (TCRs) are triggered to start signaling is still not fully understood. It has been proposed that segregation of the large membrane tyrosine phosphatase CD45 from engaged TCRs initiates signaling by favoring phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of CD3 molecules. However, whether CD45 segregation is important to initiate triggering is still uncertain. We examined CD45 segregation from TCRs engaged to anti-CD3 scFv with high or low affinity and with defined molecular lengths on glass-supported lipid bilayers using total internal reflection microscopy. Both short and elongated high-affinity anti-CD3 scFv effectively induced similar calcium mobilization, Zap70 phosphorylation, and cytokine secretion in Jurkat T cells but CD45 segregated from activated TCR microclusters significantly less for elongated versus short anti-CD3 ligands. In addition, at early times, triggering cells with both high and low affinity elongated anti-CD3 scFv resulted in similar degrees of CD3 co-localization with CD45, but only the high-affinity scFv induced T cell activation. The lack of correlation between CD45 segregation and early markers of T cell activation suggests that segregation of CD45 from engaged TCRs is not mandatory for initial triggering of TCR signaling by elongated high-affinity ligands.

A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/ 2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.

Asthma is ranked among the most common chronic conditions and has become a significant public health issue due to the recent and rapid increase in its prevalence. Investigations into the underlying genetic factors predict a heritable component for its incidence, estimated between 35% and 90% of causation. Despite the application of large-scale genome-wide association studies (GWAS) and admixture mapping approaches, the proportion of variants identified accounts for less than 15% of the observed heritability of the disease. The discrepancy between the predicted heritable component of disease and the proportion of heritability mapped to the currently identified susceptibility loci has been termed the ‘missing heritability problem.’ Here, we examine recent studies involving both the analysis of genetically encoded features that contribute to asthma and also the role of non-encoded heritable characteristics, including epigenetic, environmental, and developmental aspects of disease. The importance of vertical maternal microbiome transfer and the influence of maternal immune factors on fetal conditioning in the inheritance of disease are also discussed. In order to highlight the broad array of biological inputs that contribute to the sum of heritable risk factors associated with allergic disease incidence that, together, contribute to the induction of a pro-atopic state. Currently, there is a need to develop in-depth models of asthma risk factors to overcome the limitations encountered in the interpretation of GWAS results in isolation, which have resulted in the missing heritability problem. Hence, multiomics analyses need to be established considering genetic, epigenetic, and functional data to create a true systems biology-based approach for analyzing the regulatory pathways that underlie the inheritance of asthma and to develop accurate risk profiles for disease.

No abstract

Cytotoxic T cells (CTLs) and B cells engage distinct interactions in GVHD patients' blood and tissues, detectable in regular flow-cytometry screenings, by size and by double positive CD19-CD8 antibody markers (Deola, BMT 2017). B-CTL couplets are formed by alpha-betaTCR+ CD8+ CTLs preferentially targeting CD27+ CD19+ cells displaying an activated CD80 and CD86 phenotype. Interactions may last from 5 minutes to roughly 1 hour, and release a pattern of T cell attracting chemokines, as IP10, MIG, ITAC, which are also known GVHD biomarkers. To further unravel the mechanism of this cell interaction, we built an in-vitro model where human PBMCs cells are expanded with cognate peptides and IL2 for 1-2 weeks, then immune-selected for CD8 antigen by Miltenyi microbeads negative-selection and incubated (2-18 hours) with fresh autologous CD19-B cells, immune-selected with the same method. The interactions are studied under confocal microscope video-imaging (Zeiss LSM 880+Imaris 3D analysis software) and in flow-cytometry (SymphonyA5 BD) after deep phenotype antibody staining. The intensity of interaction, measured by fluorescence interference on cell membranes, revealed an active engagement of CD19 and CD8 antigens. CD19 antigen penetrates deeper in contacting T cells, than CD8 on B cells, and consistently with this finding, after the interactions there is an antigen exchange between cells with CD19 antigen actively transferred in CD8 cells (p value =<0.001), but not the contrary. We already proved that this type of B-T interaction is not antigen specific in CTL-to-B direction (Deola et a, JI 2008) but to exclude cross-presentation from B to CTLs and to unravel the role of CD8, we interfered by antibody blocking of MHC class I pathway on B cells and CD8 on CTLs. B-T cell interactions are not abolished after MHC-I or CD8 blocking, the intensity of coupling is unchanged after MHC-I block, and is higher after blocking CD8 (p value=<0.001). In particular, by blocking CD8 molecule, T cells target preferentially CD19+/CD27- cells rather than CD19+/27+ cells. Interestingly, B cell engagement follows 2 repetitive patterns of interaction: a high intensity interaction that visually corresponds to tight coupling cells with high CD19 penetration in T cells, and a low-intensity continuous interaction, visually measurable by cells "sniffing" each other. Both patterns correspond to diverse Calcium flux activation on T cells and B cells, suggesting functional different pathways triggered by the 2 type of interactions. Deep phenotype flow cytometry analyses after coupling reveals distinct programs triggered by the contact in both B cells and T cells. While after the interaction CTLs double their pool of perforin bearing effectors and their fraction of CD45RA-/CD27+ memory CTLs, CD19 preferentially undergo a deletion of IgD- CD27- (DN) cells (13,85%+/-1,1 and 22,95%+/-4,5 CD95/Fas+, respectively in B cells alone and B+CTLs, n=2) and a rescue of affinity mature CD27+ IgD- cells (39.8%+/-25,47 and 21,2%+/- 29% CD95/Fas+ in the same groups) CTLs are the ultimate line of "tissue attack" in GVHD and several diseases, as autoimmune diseases, cancer, viral diseases, sharing a common pathological program definable as "immune rejection". B cells are key players in immune rejection, but a link between these 2 types of cells is still unclear. Our findings enforce the hypothesis of a program of peripheral tolerance/activation triggered directly between B cells and activated CTLs in the context of inflammation and of GVHD. Disclosures No relevant conflicts of interest to declare.