A journey to your self: The vague definition of immune self and its practical implications.

The identification of immunogenic peptides has become essential in an increasing number of fields in immunology, ranging from tumor immunotherapy to vaccine development. The nature of the adaptive immune response is shaped by the similarity between foreign and self-protein sequences, a concept extensively applied in numerous studies. Can we precisely define the degree of similarity to self? Furthermore, do we accurately define immune self? In the current work, we aim to unravel the conceptual and mechanistic vagueness hindering the assessment of self-similarity. Accordingly, we demonstrate the remarkably low consistency among commonly employed measures and highlight potential avenues for future research.

Self-mediated positive selection of T cells sets an obstacle to the recognition of nonself.

Adaptive immune recognition is mediated by the binding of peptide-human leukocyte antigen complexes by T cells. Positive selection of T cells in the thymus is a fundamental step in the generation of a responding T cell repertoire: only those T cells survive that recognize human peptides presented on the surface of cortical thymic epithelial cells. We propose that while this step is essential for optimal immune function, the process results in a defective T cell repertoire because it is mediated by self-peptides. To test our hypothesis, we focused on amino acid motifs of peptides in contact with T cell receptors. We found that motifs rarely or not found in the human proteome are unlikely to be recognized by the immune system just like the ones that are not expressed in cortical thymic epithelial cells or not presented on their surface. Peptides carrying such motifs were especially dissimilar to human proteins. Importantly, we present our main findings on two independent T cell activation datasets and directly demonstrate the absence of naïve T cells in the repertoire of healthy individuals. We also show that T cell cross-reactivity is unable to compensate for the absence of positively selected T cells. Additionally, we show that the proposed mechanism could influence the risk for different infectious diseases. In sum, our results suggest a side effect of T cell positive selection, which could explain the nonresponsiveness to many nonself peptides and could improve the understanding of adaptive immune recognition.

Psoriatic Resolved Skin Epidermal Keratinocytes Retain Disease-Residual Transcriptomic and Epigenomic Profiles.

The disease-residual transcriptomic profile (DRTP) within psoriatic healed/resolved skin and epidermal tissue-resident memory T (TRM) cells have been proposed to be crucial for the recurrence of old lesions. However, it is unclear whether epidermal keratinocytes are involved in disease recurrence. There is increasing evidence regarding the importance of epigenetic mechanisms in the pathogenesis of psoriasis. Nonetheless, the epigenetic changes that contribute to the recurrence of psoriasis remain unknown. The aim of this study was to elucidate the role of keratinocytes in psoriasis relapse. The epigenetic marks 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were visualized using immunofluorescence staining, and RNA sequencing was performed on paired never-lesional and resolved epidermal and dermal compartments of skin from psoriasis patients. We observed diminished 5-mC and 5-hmC amounts and decreased mRNA expression of the ten-eleven translocation (TET) 3 enzyme in the resolved epidermis. SAMHD1, C10orf99, and AKR1B10: the highly dysregulated genes in resolved epidermis are known to be associated with pathogenesis of psoriasis, and the DRTP was enriched in WNT, TNF, and mTOR signaling pathways. Our results suggest that epigenetic changes detected in epidermal keratinocytes of resolved skin may be responsible for the DRTP in the same regions. Thus, the DRTP of keratinocytes may contribute to site-specific local relapse.

Pathogen diversity drives the evolution of generalist MHC-II alleles in human populations.

Central players of the adaptive immune system are the groups of proteins encoded in the major histocompatibility complex (MHC), which shape the immune response against pathogens and tolerance to self-peptides. The corresponding genomic region is of particular interest, as it harbors more disease associations than any other region in the human genome, including associations with infectious diseases, autoimmune disorders, cancers, and neuropsychiatric diseases. Certain MHC molecules can bind to a much wider range of epitopes than others, but the functional implication of such an elevated epitope-binding repertoire has remained largely unclear. It has been suggested that by recognizing more peptide segments, such promiscuous MHC molecules promote immune response against a broader range of pathogens. If so, the geographical distribution of MHC promiscuity level should be shaped by pathogen diversity. Three lines of evidence support the hypothesis. First, we found that in pathogen-rich geographical regions, humans are more likely to carry highly promiscuous MHC class II DRB1 alleles. Second, the switch between specialist and generalist antigen presentation has occurred repeatedly and in a rapid manner during human evolution. Third, molecular positions that define promiscuity level of MHC class II molecules are especially diverse and are under positive selection in human populations. Taken together, our work indicates that pathogen load maintains generalist adaptive immune recognition, with implications for medical genetics and epidemiology.

Differential role of D cyclins in the regulation of cell cycle by influencing Ki67 expression in HaCaT cells.

D-type cyclins are important regulatory proteins of the G1/S phase of the cell cycle however, their specific functions are only partially understood. We show that silencing of individual D-type cyclins has no effect on the proliferation and morphology of Immortalized non-tumorigenic human epidermal (HaCaT) cells, while double and triple D cyclin silencing results in the failure of the cytokinesis leading to the appearance of large multinucleated cells. Both CDC20 and Ki67 mRNA is downregulated in these cells. Ki67 mRNA silenced cells show similar multinucleated cellular phenotype as double or triple D cyclin silenced cells without affecting D cyclin expression, suggesting that Ki67 is necessary for normal G2/M transition. Our data have revealed that cyclin D1 may have a leading role in G1/S phase regulation and suggest an incomplete functional overlap among D cyclins. Our results indicate that beside their well-known functions during the G0-G1/S phase, D-type cyclins play a pivotal role in the regulation of mitosis via influencing Ki67 expression in a downstream manner probably through E2F1 activation in HaCaT cells.

Switching on RNA silencing suppressor activity by restoring argonaute binding to a viral protein.

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor.

Ghrelin and nicotine stimulate equally the dopamine release in the rat amygdala.

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward processes and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Ghrelin and nicotine activates the mesolimbicocortical dopaminergic pathways via growth hormone secretagogue receptors (GHS-R1A) and nicotinic acetylcholine receptors (nAchR), respectively, resulting in the release of dopamine in the nucleus accumbens, the amygdala and the prefrontal cortex. In the present study an in vitro superfusion of rat amygdalar slices was performed in order to investigate the direct action of ghrelin and nicotine on the amygdalar dopamine release. Ghrelin increased significantly the dopamine release from the rat amygdala following electrical stimulation. This effect was inhibited by both the selective GHS-R1A antagonist GHRP-6 and the selective nAchR antagonist mecamylamine. Under the same conditions, nicotine also increased significantly the dopamine release from the rat amygdala. This effect was antagonized by mecamylamine, but not by GHRP-6. Co-administration of ghrelin and nicotine induced a similar increase of amygdalar dopamine release. This stimulatory effect was partially reversed by both GHRP-6 and mecamylamine. The present results demonstrate that both ghrelin and nicotine stimulates directly the dopamine release in the amygdala, an important dopaminergic target area of the mesolimbicocortical pathway.

Unraveling Transcriptome Profile, Epigenetic Dynamics, and Morphological Changes in Psoriasis-like Keratinocytes: "Insights into Similarity with Psoriatic Lesional Epidermis".

Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

Single-cell immunophenotyping revealed the association of CD4+ central and CD4+ effector memory T cells linking exacerbating chronic obstructive pulmonary disease and NSCLC.

Introduction: Tobacco smoking generates airway inflammation in chronic obstructive pulmonary disease (COPD), and its involvement in the development of lung cancer is still among the leading causes of early death. Therefore, we aimed to have a better understanding of the disbalance in immunoregulation in chronic inflammatory conditions in smoker subjects with stable COPD (stCOPD), exacerbating COPD (exCOPD), or non-small cell lung cancer (NSCLC). Methods: Smoker controls without chronic illness were recruited as controls. Through extensive mapping of single cells, surface receptor quantification was achieved by single-cell mass cytometry (CyTOF) with 29 antibodies. The CyTOF characterized 14 main immune subsets such as CD4+, CD8+, CD4+/CD8+, CD4-/CD8-, and γ/δ T cells and other subsets such as CD4+ or CD8+ NKT cells, NK cells, B cells, plasmablasts, monocytes, CD11cdim, mDCs, and pDCs. The CD4+ central memory (CM) T cells (CD4+/CD45RA-/CD45RO+/CD197+) and CD4+ effector memory (EM) T cells (CD4+/CD45RA-/CD45RO+/CD197-) were FACS-sorted for RNA-Seq analysis. Plasma samples were assayed by Luminex MAGPIX® for the quantitative measurement of 17 soluble immuno-oncology mediators (BTLA, CD28, CD80, CD27, CD40, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2) in the four studied groups. Results: Our focus was on T-cell-dependent differences in COPD and NSCLC, where peripheral CD4+ central memory and CD4+ effector memory cells showed a significant reduction in exCOPD and CD4+ CM showed elevation in NSCLC. The transcriptome analysis delineated a perfect correlation of differentially expressed genes between exacerbating COPD and NSCLC-derived peripheral CD4+ CM or CD4+ EM cells. The measurement of 17 immuno-oncology soluble mediators revealed a disease-associated phenotype in the peripheral blood of stCOPD, exCOPD, and NSCLC patients. Discussion: The applied single-cell mass cytometry, the whole transcriptome profiling of peripheral CD4+ memory cells, and the quantification of 17 plasma mediators provided complex data that may contribute to the understanding of the disbalance in immune homeostasis generated or sustained by tobacco smoking in COPD and NSCLC.

Phenotypic plasticity of melanocytes derived from human adult skin.

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP-1 and c-Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation.

Negative trade-off between neoantigen repertoire breadth and the specificity of HLA-I molecules shapes antitumor immunity.

Human leukocyte antigen class I (HLA-I) genes shape our immune response against pathogens and cancer. Certain HLA-I variants can bind a wider range of peptides than others, a feature that could be favorable against a range of viral diseases. However, the implications of this phenomenon on cancer immune response are unknown. Here we quantified peptide repertoire breadth (or promiscuity) of a representative set of HLA-I alleles and found that patients with cancer who were carrying HLA-I alleles with high peptide-binding promiscuity have significantly worse prognosis after immune checkpoint inhibition. This can be explained by a reduced capacity of the immune system to discriminate tumor neopeptides from self-peptides when patients carry highly promiscuous HLA-I variants, shifting the regulation of tumor-infiltrating T cells from activation to tolerance. In summary, HLA-I peptide-binding specificity shapes neopeptide immunogenicity and the self-immunopeptidome repertoire in an antagonistic manner, and could underlie a negative trade-off between antitumor immunity and genetic susceptibility to viral infections.

Comprehensive Proteomic Analysis Reveals Intermediate Stage of Non-Lesional Psoriatic Skin and Points out the Importance of Proteins Outside this Trend.

To better understand the pathomechanism of psoriasis, a comparative proteomic analysis was performed with non-lesional and lesional skin from psoriasis patients and skin from healthy individuals. Strikingly, 79.9% of the proteins that were differentially expressed in lesional and healthy skin exhibited expression levels in non-lesional skin that were within twofold of the levels observed in healthy and lesional skin, suggesting that non-lesional skin represents an intermediate stage. Proteins outside this trend were categorized into three groups: I. proteins in non-lesional skin exhibiting expression similar to lesional skin, which might be predisposing factors (i.e., CSE1L, GART, MYO18A and UGDH); II. proteins that were differentially expressed in non-lesional and lesional skin but not in healthy and lesional skin, which might be non-lesional characteristic alteration (i.e., CHCHD6, CHMP5, FLOT2, ITGA7, LEMD2, NOP56, PLVAP and RRAS); and III. proteins with contrasting differential expression in non-lesional and lesional skin compared to healthy skin, which might contribute to maintaining the non-lesional state (i.e., ITGA7, ITGA8, PLVAP, PSAPL1, SMARCA5 and XP32). Finally, proteins differentially expressed in lesions may indicate increased sensitivity to stimuli, peripheral nervous system alterations, furthermore MYBBP1A and PRKDC were identified as potential regulators of key pathomechanisms, including stress and immune response, proliferation and differentiation.

Peptide presentation by HLA-DQ molecules is associated with the development of immune tolerance.

HLA class II proteins are important elements of human adaptive immune recognition and are associated with numerous infectious and immune-mediated diseases. These highly variable molecules can be classified into DP, DQ and DR groups. It has been proposed that in contrast with DP and DR, epitope binding by DQ variants rather results in immune tolerance. However, the pieces of evidence are limited and controversial. We found that DQ molecules bind more human epitopes than DR. Pathogen-associated epitopes bound by DQ molecules are more similar to human proteins than the ones bound by DR. Accordingly, DQ molecules bind epitopes of significantly different pathogen species. Moreover, the binding of autoimmunity-associated epitopes by DQ confers protection from autoimmune diseases. Our results suggest a special role of HLA-DQ in immune homeostasis and help to better understand the association of HLA molecules with infectious and autoimmune diseases.

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro.

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis.

Novel factors in the pathogenesis of psoriasis and potential drug candidates are found with systems biology approach.

Psoriasis is a multifactorial inflammatory skin disease characterized by increased proliferation of keratinocytes, activation of immune cells and susceptibility to metabolic syndrome. Systems biology approach makes it possible to reveal novel important factors in the pathogenesis of the disease. Protein-protein, protein-DNA, merged (containing both protein-protein and protein-DNA interactions) and chemical-protein interaction networks were constructed consisting of differentially expressed genes (DEG) between lesional and non-lesional skin samples of psoriatic patients and/or the encoded proteins. DEGs were determined by microarray meta-analysis using MetaOMICS package. We used STRING for protein-protein, CisRED for protein-DNA and STITCH for chemical-protein interaction network construction. General network-, cluster- and motif-analysis were carried out in each network. Many DEG-coded proteins (CCNA2, FYN, PIK3R1, CTGF, F3) and transcription factors (AR, TFDP1, MEF2A, MECOM) were identified as central nodes, suggesting their potential role in psoriasis pathogenesis. CCNA2, TFDP1 and MECOM might play role in the hyperproliferation of keratinocytes, whereas FYN may be involved in the disturbed immunity in psoriasis. AR can be an important link between inflammation and insulin resistance, while MEF2A has role in insulin signaling. A controller sub-network was constructed from interlinked positive feedback loops that with the capability to maintain psoriatic lesional phenotype. Analysis of chemical-protein interaction networks detected 34 drugs with previously confirmed disease-modifying effects, 23 drugs with some experimental evidences, and 21 drugs with case reports suggesting their positive or negative effects. In addition, 99 unpublished drug candidates were also found, that might serve future treatments for psoriasis.

Ghrelin amplifies the nicotine-induced dopamine release in the rat striatum.

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward mechanisms and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Nicotine activates the mesencephalic dopaminergic neurons via nicotinic acetylcholine receptors (nAchR). Ghrelin stimulates the dopaminergic neurons via growth hormone secretagogue receptors (GHS-R1A) in the ventral tegmental area and the substantia nigra pars compacta resulting in the release of dopamine in the ventral and dorsal striatum, respectively. In the present study an in vitro superfusion of rat striatal slices was performed, in order to investigate the direct action of ghrelin on the striatal dopamine release and the interaction of ghrelin with nicotine through this neurotransmitter release. Ghrelin increased significantly the dopamine release from the rat striatum following electrical stimulation. This stimulatory effect was reversed by both the selective nAchR antagonist mecamylamine and the selective GHS-R1A antagonist GHRP-6. Nicotine also increased significantly the dopamine release under the same conditions. This stimulatory effect was antagonized by mecamylamine, but not by GHRP-6. Ghrelin further stimulated the nicotine-induced dopamine release and this effect was abolished by mecamylamine and was partially inhibited by GHRP-6. The present results demonstrate that ghrelin stimulates directly the dopamine release and amplifies the nicotine-induced dopamine release in the rat striatum. We presume that striatal cholinergic interneurons also express GHS-R1A, through which ghrelin can amplify the nicotine-induced dopamine release in the striatum. This study provides further evidence of the impact of ghrelin on the mesolimbic and nigrostriatal dopaminergic pathways. It also suggests that ghrelin signaling may serve as a novel pharmacological target for treatment of addictive and neurodegenerative disorders.

Intranasal application of secretin, similarly to intracerebroventricular administration, influences the motor behavior of mice probably through specific receptors.

Secretin and its receptors show wide distribution in the central nervous system. It was demonstrated previously that intravenous (i.v.) and intracerebroventricular (i.c.v.) application of secretin influenced the behavior of rat, mouse, and human. In our previous experiment, we used a special animal model, Japanese waltzing mice (JWM). These animals run around without stopping (the ambulation distance is very limited) and they do not bother with their environment. The i.c.v. secretin attenuated this hyperactive repetitive movement. In the present work, the effect of i.c.v. and intranasal (i.n.) application of secretin was compared. We have also looked for the presence of secretin receptors in the brain structures related to motor functions. Two micrograms of i.c.v. secretin improved the horizontal movement of JWM, enhancing the ambulation distance. It was nearly threefold higher in treated than in control animals. The i.n. application of secretin to the left nostril once or twice a day or once for 3 days more effectively enhanced the ambulation distance than i.c.v. administration. When secretin was given twice a day for 3 days it had no effect. Secretin did not improve the explorative behavior (the rearing), of JWM. With the use of in situ hybridization, we have found very dense secretin receptor labeling in the cerebellum. In the primary motor cortex and in the striatum, only a few labeled cells were seen. It was supposed that secretin exerted its effect through specific receptors, mainly present in the cerebellum.