Enhancing Regulatory T Cells to Treat Inflammatory and Autoimmune Diseases.

Regulatory T cells (Tregs) control immune responses and are essential to maintain immune homeostasis and self-tolerance. Hence, it is no coincidence that autoimmune and chronic inflammatory disorders are associated with defects in Tregs. These diseases have currently no cure and are treated with palliative drugs such as immunosuppressant and immunomodulatory agents. Thereby, there is a great interest in developing medical interventions against these diseases based on enhancing Treg cell function and numbers. Here, we give an overview of Treg cell ontogeny and function, paying particular attention to mucosal Tregs. We review some notable approaches to enhance immunomodulation by Tregs with therapeutic purposes including adoptive Treg cell transfer therapy and discuss relevant clinical trials for inflammatory bowel disease. We next introduce ways to expand mucosal Tregs in vivo using microbiota and dietary products that have been the focus of clinical trials in various autoimmune and chronic-inflammatory diseases.

Human rhinovirus-specific CD8 T cell responses target conserved and unusual epitopes.

Human Rhinovirus (HRV) is a major cause of common cold, bronchiolitis, and exacerbations of chronic pulmonary diseases such as asthma. CD8 T cell responses likely play an important role in the control of HRV infection but, surprisingly, HRV-specific CD8 T cell epitopes remain yet to be identified. Here, we approached the discovery and characterization of conserved HRV-specific CD8 T cell epitopes from species A (HRV A) and C (HRV C), the most frequent subtypes in the clinics of various pulmonary diseases. We found IFNγ-ELISPOT positive responses to 23 conserved HRV-specific peptides on peripheral blood mononuclear cells (PBMCs) from 14 HLA I typed subjects. Peptide-specific IFNγ production by CD8 T cells and binding to the relevant HLA I were confirmed for six HRV A-specific and three HRV C-specific CD8 T cell epitopes. In addition, we validated A*02:01-restricted epitopes by DimerX staining and found out that these peptides mediated cytotoxicity. All these A*02:01-restricted epitopes were 9-mers but, interestingly, we also identified and validated an unusually long 16-mer epitope peptide restricted by A*02:01, HRVC1791-1806 (GLEPLDLNTSAGFPYV). HRV-specific CD8 T cell epitopes describe here are expected to elicit CD8 T cell responses in up to 87% of the population and could be key for developing an HRV vaccine.

Immune Tolerance in the Oral Mucosa.

The oral mucosa is a site of intense immune activity, where a large variety of immune cells meet to provide a first line of defense against pathogenic organisms. Interestingly, the oral mucosa is exposed to a plethora of antigens from food and commensal bacteria that must be tolerated. The mechanisms that enable this tolerance are not yet fully defined. Many works have focused on active immune mechanisms involving dendritic and regulatory T cells. However, epithelial cells also make a major contribution to tolerance by influencing both innate and adaptive immunity. Therefore, the tolerogenic mechanisms concurring in the oral mucosa are intertwined. Here, we review them systematically, paying special attention to the role of oral epithelial cells.

Cellular Integrin α5ß1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells.

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size-exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5ß1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.

Identification of CD8+ T cell epitopes through proteasome cleavage site predictions.

BACKGROUND: We previously introduced PCPS (Proteasome Cleavage Prediction Server), a web-based tool to predict proteasome cleavage sites using n-grams. Here, we evaluated the ability of PCPS immunoproteasome cleavage model to discriminate CD8+ T cell epitopes. RESULTS: We first assembled an epitope dataset consisting of 844 unique virus-specific CD8+ T cell epitopes and their source proteins. We then analyzed cleavage predictions by PCPS immunoproteasome cleavage model on this dataset and compared them with those provided by a related method implemented by NetChop web server. PCPS was clearly superior to NetChop in term of sensitivity (0.89 vs. 0.79) but somewhat inferior with regard to specificity (0.55 vs. 0.60). Judging by the Mathew's Correlation Coefficient, PCPS predictions were overall superior to those provided by NetChop (0.46 vs. 0.39). We next analyzed the power of C-terminal cleavage predictions provided by the same PCPS model to discriminate CD8+ T cell epitopes, finding that they could be discriminated from random peptides with an accuracy of 0.74. Following these results, we tuned the PCPS web server to predict CD8+ T cell epitopes and predicted the entire SARS-CoV-2 epitope space. CONCLUSIONS: We report an improved version of PCPS named iPCPS for predicting proteasome cleavage sites and peptides with CD8+ T cell epitope features. iPCPS is available for free public use at https://imed.med.ucm.es/Tools/pcps/ .

Correction to: Computational assembly of a human Cytomegalovirus vaccine upon experimental epitope legacy.

After publication of the original article [1], we were notified that legends of Fig. 1 and Fig. 2 have been swapped.

Computational assembly of a human Cytomegalovirus vaccine upon experimental epitope legacy.

BACKGROUND: Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus affecting approximately 90% of the world population. HCMV causes disease in immunologically naive and immunosuppressed patients. The prevention, diagnosis and therapy of HCMV infection are thus crucial to public health. The availability of effective prophylactic and therapeutic treatments remain a significant challenge and no vaccine is currently available. Here, we sought to define an epitope-based vaccine against HCMV, eliciting B and T cell responses, from experimentally defined HCMV-specific epitopes. RESULTS: We selected 398 and 790 experimentally validated HCMV-specific B and T cell epitopes, respectively, from available epitope resources and apply a knowledge-based approach in combination with immunoinformatic predictions to ensemble a universal vaccine against HCMV. The T cell component consists of 6 CD8 and 6 CD4 T cell epitopes that are conserved among HCMV strains. All CD8 T cell epitopes were reported to induce cytotoxic activity, are derived from early expressed genes and are predicted to provide population protection coverage over 97%. The CD4 T cell epitopes are derived from HCMV structural proteins and provide a population protection coverage over 92%. The B cell component consists of just 3 B cell epitopes from the ectodomain of glycoproteins L and H that are highly flexible and exposed to the solvent. CONCLUSIONS: We have defined a multiantigenic epitope vaccine ensemble against the HCMV that should elicit T and B cell responses in the entire population. Importantly, although we arrived to this epitope ensemble with the help of computational predictions, the actual epitopes are not predicted but are known to be immunogenic.

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis.

Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2'-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß2 integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

Selection of conserved epitopes from hepatitis C virus for pan-populational stimulation of T-cell responses.

The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server.

EPIPOX: A Resource Facilitating Epitope-Vaccine Design Against Human Pathogenic Orthopoxviruses.

EPIPOX is a specialized online resource intended to facilitate the design of epitope-based vaccines against orthopoxviruses. EPIPOX is built upon a collection of T cell epitopes that are shared by eight pathogenic orthopoxviruses, including variola minor and major strains, monkeypox, cowpox, and vaccinia viruses. In EPIPOX, users can select T cell epitopes attending to the predicted binding to distinct major histocompatibility molecules (MHC) and according to various features that may have an impact on epitope immunogenicity. Among others, EPIPOX allows to discern epitopes by their structural location in the virion and the temporal expression of the counterpart antigens. Overall, the annotations in EPIPOX are optimized to facilitate the rational design of T cell epitope-based vaccines. In this chapter, we describe the main features of EPIPOX and exemplify its use, retrieving orthopoxvirus-specific T cell epitopes with features set to enhance their immunogenicity. EPIPOX is available for free public use at http://bio.med.ucm.es/epipox/ .

Combining different bacteria in vaccine formulations enhances the chance for antiviral cross-reactive immunity: a detailed in silico analysis for influenza A virus.

Bacteria are well known to provide heterologous immunity against viral infections through various mechanisms including the induction of innate trained immunity and adaptive cross-reactive immunity. Cross-reactive immunity from bacteria to viruses is responsible for long-term protection and yet its role has been downplayed due the difficulty of determining antigen-specific responses. Here, we carried out a systematic evaluation of the potential cross-reactive immunity from selected bacteria known to induce heterologous immunity against various viruses causing recurrent respiratory infections. The bacteria selected in this work were Bacillus Calmette Guerin and those included in the poly-bacterial preparation MV130: Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Klebisella pneumoniae, Branhamella catarrhalis and Haemophilus influenzae. The virus included influenza A and B viruses, human rhinovirus A, B and C, respiratory syncytial virus A and B and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through BLAST searches, we first identified the shared peptidome space (identity ≥ 80%, in at least 8 residues) between bacteria and viruses, and subsequently predicted T and B cell epitopes within shared peptides. Interestingly, the potential epitope spaces shared between bacteria in MV130 and viruses are non-overlapping. Hence, combining diverse bacteria can enhance cross-reactive immunity. We next analyzed in detail the cross-reactive T and B cell epitopes between MV130 and influenza A virus. We found that MV130 contains numerous cross-reactive T cell epitopes with high population protection coverage and potentially neutralizing B cell epitopes recognizing hemagglutinin and matrix protein 2. These results contribute to explain the immune enhancing properties of MV130 observed in the clinic against respiratory viral infections.

Corrigendum: Combining different bacteria in vaccine formulations enhances the chance for antiviral cross-reactive immunity: a detailed in silico analysis for influenza A virus.

[This corrects the article DOI: 10.3389/fimmu.2023.1235053.].

Rap1-GTP-interacting adaptor molecule (RIAM) protein controls invasion and growth of melanoma cells.

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces ß1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.

The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9.

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.

Expression of the phagocytic receptors αMß2 and αXß2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMß2, CD11b/CD18) and CR4 (αXß2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to ß2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (ß2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMß2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMß2 and αXß2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Structural, biochemical, and functional properties of the Rap1-Interacting Adaptor Molecule (RIAM).

Leukocytes, the leading players of immune system, are involved in innate and adaptive immune responses. Leukocyte adhesion to endothelial cells during transmigration or to antigen presenting cells during T cell activation, requires integrin activation through a process termed inside-out integrin signaling. In hematopoietic cells, Rap1 and its downstream effector RIAM (Rap1-interacting adaptor molecule) form a cornerstone for inside-out integrin activation. The Rap1/RIAM pathway is involved in signal integration for activation, actin remodeling and cytoskeletal reorganization in T cells, as well as in myeloid cell differentiation and function. RIAM is instrumental for phagocytosis, a process requiring particle recognition, cytoskeletal remodeling and membrane protrusion for engulfment and digestion. In the present review, we discuss the structural and molecular properties of RIAM and the recent discoveries regarding the functional role of the Rap1/RIAM module in hematopoietic cells.

BCEPS: A Web Server to Predict Linear B Cell Epitopes with Enhanced Immunogenicity and Cross-Reactivity.

Prediction of linear B cell epitopes is of interest for the production of antigen-specific antibodies and the design of peptide-based vaccines. Here, we present BCEPS, a web server for predicting linear B cell epitopes tailored to select epitopes that are immunogenic and capable of inducing cross-reactive antibodies with native antigens. BCEPS implements various machine learning models trained on a dataset including 555 linearized conformational B cell epitopes that were mined from antibody-antigen protein structures. The best performing model, based on a support vector machine, reached an accuracy of 75.38% ± 5.02. In an independent dataset consisting of B cell epitopes retrieved from the Immune Epitope Database (IEDB), this model achieved an accuracy of 67.05%. In BCEPS, predicted epitopes can be ranked according to properties such as flexibility, accessibility and hydrophilicity, and with regard to immunogenicity, as judged by their predicted presentation by MHC II molecules. BCEPS also detects if predicted epitopes are located in ectodomains of membrane proteins and if they possess N-glycosylation sites hindering antibody recognition. Finally, we exemplified the use of BCEPS in the SARS-CoV-2 Spike protein, showing that it can identify B cell epitopes targeted by neutralizing antibodies.

Human Oral Epithelial Cells Suppress T Cell Function via Prostaglandin E2 Secretion.

The oral mucosa is constantly exposed to a plethora of stimuli including food antigens, commensal microbiota and pathogens, requiring distinct immune responses. We previously reported that human oral epithelial cells (OECs) suppress immune responses to bacteria, using H413 and TR146 OEC lines and primary OECs in co-culture with dendritic cells (DCs) and T cells (OEC-conditioned cells). OECs reduced DCs expression of CD80/CD86 and IL-12/TNFα release and impaired T cell activation. Here, we further evaluated the immunosuppression by these OECs and investigated the underlying mechanisms. OEC-conditioned DCs did not induce CD4 T cell polarization towards Treg, judging by the absence of FoxP3 expression. OECs also repressed T-bet/IFNγ expression in CD4 and CD8 T cells activated by DCs or anti-CD3/CD28 antibodies. This inhibition depended on OEC:T cell ratio and IFNγ repression occurred at the transcriptional level. Time-lapse experiments showed that OECs inhibited early steps of T cell activation, consistent with OECs inability to suppress T cells stimulated with PMA/ionomycin. Blocking CD40/CD40L, CD58/CD2 and PD-L1/PD-1 interactions with specific antibodies did not disrupt T cell suppression by OECs. However, preventing prostaglandin E2 (PGE2) synthesis or blocking PGE2 binding to the cognate EP2/EP4 receptors, restored IFNγ and TNFα production in OEC-conditioned T cells. Finally, treating OECs with poly(I:C), which simulates viral infections, limited T cell suppression. Overall, these results point to an inherent ability of OECs to suppress immune responses, which can nonetheless be eluded when OECs are under direct assault.

Functional Integrin Regulation Through Interactions with Tetraspanin CD9.

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.