Expression Of Antigen-presenting Molecules Research Articles

Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation.

The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells). Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout. Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells. This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.

Microneedle Delivery of an Adjuvanted Microparticulate Vaccine Induces High Antibody Levels in Mice Vaccinated against Coronavirus.

This ‘proof-of-concept’ study aimed to test the microparticulate vaccine delivery system and a transdermal vaccine administration strategy using dissolving microneedles (MN). For this purpose, we formulated poly(lactic-co-glycolic) acid (PLGA) microparticles (MP) encapsulating the inactivated canine coronavirus (iCCoV), as a model antigen, along with adjuvant MP encapsulating Alhydrogel® and AddaVax. We characterized the vaccine MP for size, surface charge, morphology, and encapsulation efficiency. Further, we evaluated the in vitro immunogenicity, cytotoxicity, and antigen-presentation of vaccine/adjuvant MP in murine dendritic cells (DCs). Additionally, we tested the in vivo immunogenicity of the MP vaccine in mice through MN administration. We evaluated the serum IgG, IgA, IgG1, and IgG2a responses using an enzyme-linked immunosorbent assay. The results indicate that the particulate form of the vaccine is more immunogenic than the antigen suspension in vitro. We found the vaccine/adjuvant MP to be non-cytotoxic to DCs. The expression of antigen-presenting molecules, MHC I/II, and their costimulatory molecules, CD80/40, increased with the addition of the adjuvants. Moreover, the results suggest that the MP vaccine is cross presented by the DCs. In vivo, the adjuvanted MP vaccine induced increased antibody levels in mice following vaccination and will further be assessed for its cell-mediated responses.

Enhanced Immunogenicity of an Influenza Ectodomain Matrix-2 Protein Virus-like Particle (M2e VLP) Using Polymeric Microparticles for Vaccine Delivery.

In this study, we demonstrate how encapsulating a conserved influenza ectodomain matrix-2 protein virus-like particle (M2e5x VLP) into a pre-crosslinked bovine serum albumin (BSA) polymeric matrix enhances in vitro antigen immunogenicity and in vivo efficacy. The spray-dried M2e5x VLP-loaded BSA microparticles (MPs) showed enhanced stimulation of antigen presenting cells (APCs), as confirmed through nitrite production and increased antigen–cell interactions seen in real time using live-cell imaging. Next, to further boost the immunogenicity of M2e5x VLP microparticles, M2e5x MPs were combined with Alhydrogel® and monophosphoryl lipid-A (MPL-A®) adjuvant microparticles. M2e5x VLP MPs and the combination VLP M2e5x VLP + Alhydrogel® + MPL-A® MPs elicited a significant increase in the expression of antigen-presenting molecules in dendritic cells compared to M2e5x VLP alone. Lastly, for preliminary evaluation of in vivo efficacy, the vaccine was administered in mice through the skin using an ablative laser. The M2e5x VLP + Alhydrogel® + MPL-A® MPs were shown to induce high levels of M2e-specific IgG antibodies. Further, a challenge with live influenza revealed heightened T-cell stimulation in immune organs of mice immunized with M2e5x VLP + Alhydrogel® + MPL-A® MPs. Hence, we utilized the advantages of both VLP and polymeric delivery platforms to enhance antigen immunogenicity and adaptive immunity in vivo.

THU191 - Ebola virus infection promotes reduced gene expression of antigen presentation molecules in hepatic CD68+ macrophages in cynomolgus macaques

THU191 - Ebola virus infection promotes reduced gene expression of antigen presentation molecules in hepatic CD68+ macrophages in cynomolgus macaques

MHC Class I Stability is Modulated by Cell Surface Sialylation in Human Dendritic Cells.

Maturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to identify sialylated proteins at DC surface and comprehend their role and modulation. Mass spectrometry analysis of DC’s proteins, pulled down by a sialic acid binding lectin, identified molecules of the major human histocompatibility complex class I (MHC-I), known as human leucocyte antigen (HLA). After desialylation, DCs showed significantly higher reactivity with antibodies specific for properly folded MHC-I-β2-microglobulin complex and for β2-microglobulin but showed significant lower reactivity with an antibody specific for free MHC-I heavy chain. Similar results for antibody reactivities were observed for TAP2-deficient lymphoblastoid T2 cells, which express HLA-A*02:01. Using fluorescent peptide specifically fitting the groove of HLA-A*02:01, instead of antibody staining, also showed higher peptide binding on desialylated cells, confirming higher surface expression of MHC-I complex. A decay assay showed that desialylation doubled the half-life of MHC-I molecules at cell surface in both DCs and T2 cells. The biological impact of DC´s desialylation was evaluated in co-cultures with autologous T cells, showing higher number and earlier immunological synapses, and consequent significantly increased production of IFN-γ by T cells. In summary, sialic acid content modulates the expression and stability of complex MHC-I, which may account for the improved DC-T synapses.

Combining Whole Exome Sequencing and Rnaseq to Provide a Comprehensive Landscape of the Mechanisms of Post-Transplantation Leukemia Relapse

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents an effective treatment for many hematological malignancies, but post-transplantation relapses remain frequent, and their biological bases poorly understood.Here we combined Whole Exome Sequencing (WES) and RNA-Seq to compare the features of 15 cases of Acute Myeloid Leukemia before allo-HSCT and at post-transplantation relapse. Leukemic blasts were collected and purified at the two timepoints, and compared between each other and with patient and donor germline controls. Three donors were HLA-identical siblings, 7 were matched unrelated volunteers (all of which were matched to the patient for 10/10 HLA loci) and 5 were HLA-haploidentical relatives. Median time from allo-HSCT to relapse was 144 days (range 33-574).The average coverage for WES, consistent between all cases, was 120x for leukemia samples and 60X for germline controls.Analysis of copy number alterations did not reveal major genomic alteration acquired at relapse, except for one patient who gained trisomy for chr. 6 and 21.Also the transitions/transversions ratio remained constant between the pre-Tx and post-Tx samples.We detected an average of 15 somatic variants (SNV and small InDel) per leukemia sample, and the overall mutational burden increased significantly between pre- and post-Tx samples (Wilcoxon test, pvalue = 0.0088). We evidenced 4 major patterns of clonal evolution. In pattern 1 (n=3), the pre-Tx clones persisted unchanged at relapse, in pattern 2 (n=6) and 3 (n=2), subclones were gained or lost, respectively, whereas in pattern 4 (n=4) we found a mixed scenario.Sixty somatic variants were present only in relapsed samples, encompassing known AML driver genes, including KRAS and WT1 (de novo mutated at relapse in 2 patients). Unexpectedly, WES analysis did not detect relapse-specific mutations in genes related to immune function, and even an ad hoc developed pipeline for the analysis of somatic mutations in HLA class I and class II genes did not detect any denovo acquired sequence abnormality.Conversely, a linear model analysis of RNA-seq showed ~800 genes significantly deregulated in AML blasts at post-transplantation relapse. The down-regulated genes were mainly immune-related, encompassing in particular those involved in HLA class II antigen presentation. Upregulated genes comprised genes relevant to DNA replication and cell cycle control (including several component of Minichromosome Maintanance Complex). Of interest, these two processes appeared also to cluster independently in our patient series, suggesting the presence of different transcriptional mechanisms of relapse.Of interest, we observed HLA class II downregulation also in several cases in which donor and patient were matched for those loci. Thus, to understand whether the decreased expression of antigen presentation molecules could in these cases be driven by an increase in the levels of presented antigens, we extracted from the RNA-seq dataset information regarding known leukemia associated antigens, finding several of them upregulated at post-transplantation relapse (MPO, TERT, PRTN3)Moreover, we combined WES and RNA-seq data to predict the number of patient-specific neoantigens and minor histocompatibility antigens (MiHAgs) presented on leukemia blast before and after allo-HSCT. In line with the low overall burden of mutations, we predicted a very low number of neoantigens per case (on average 3 per sample, ranging from 0 to 20), increasing at relapse in 5/15 patients. The number of predicted MiHAgs was sizably higher, and varied considerably in relation to donor-recipient matching (on average 481 in haploidentical HSCTs, 874 in HSCTs from HLA-identical siblings, and 1435 in unrelated donor HSCTs). However, the overall level of expression of MiHAgs did not vary between pre- and post-Tx samplesOur results provide for the first time a detailed landscape of the many features that shape the interplay between immune system and leukemia in allo-HSCT. The resulting picture is composite, suggesting that mechanisms of relapse are highly patient-specific and combine genomic and non-genomic, immunological and non-immunological changes. For this reason we modeled our data in a comprehensive framework that we termed “relapsogram”, that might help in elucidating the peculiarity of each case of relapse and in customizing the therapy. DisclosuresStoelzel:Neovii: Speakers Bureau. Bonini:Intellia Therapeutics: Research Funding. Vago:Moderna TX: Research Funding; GENDX: Research Funding.

TLR-induced secretion of novel cytokine IL-27 is defective in newly diagnosed type-2 diabetic subjects

Toll-like receptors (TLRs), the innate immune receptors, act as sentinels bridging both innate and adaptive arms of immunity. In the present study, we estimated TLR-induced secretion of IL-27, IL-12, IL-23, IL-8, IP-10, IL-17, IL-6 and TNF-α (by ELISA) and expression of Human Leukocyte Antigen- (Human Leukocyte Antigen - antigen D Related (HLA-DR), CD69, CD80 (also known asB7-1) (by flowcytometry) and Activating Transcription Factor 3(ATF3) (by qRT-PCR) in whole blood cultures of control and type-2 diabetic (both newly diagnosed/NDD and known/KDM) subjects. TLR-induced secretion of IL-27 was significantly reduced in the NDD group compared to the control (Normal Glucose Tolerance (NGT)) and KDM groups. On the other hand, the expression of CD80 was significantly upregulated in both the monocytes and B cells in KDM group. This was associated with increased T cell activation (CD3+CD69+HLA-DR+) with increased IL-17 and reduced TNF-α secretion in this group. Impaired TLR-induced IL-27 secretion and augmented expression of antigen presentation molecules result in chronic T cell activation which may fuel T cell-mediated inflammation in type-2 diabetes.

HTLV-1-infected thymic epithelial cells convey the virus to CD4+ T lymphocytes

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell–cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4+ T cell line and to CD4+ T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4+ T lymphocytes, which in turn will cause disease in the periphery.

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice

ObjectiveType 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. MethodsTransgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks. ResultsIn transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. ConclusionsLocal expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.

Norovirus antagonism of B-cell antigen presentation results in impaired control of acute infection.

Human noroviruses are a leading cause of gastroenteritis so vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen presenting cells, we tested whether noroviruses regulate B cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8+ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8+ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for activation of this pathway and ultimately only B cells were required for clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8+ T cell responses.

Bjcul, a snake venom lectin, modulates monocyte-derived macrophages to a pro-inflammatory profile in vitro

Macrophages are cells of high plasticity and can act in different ways to ensure that the appropriate immune response remains controlled. This study shows the effects of the C-type Bothrops jararacussu venom lectin (BJcuL) on the activation of human macrophages derived from the U937 cell line. BJcuL binds on the cell surface, and this event is inhibited by its specific carbohydrate. It induced phagocytosis and production of H2O2, and expression of antigen presentation molecules. It also enhanced the production of TNF-α, GM-CSF and IL-6 by macrophages and indirectly induced T cells to an increased production of TNF-α, IFN-γ and IL-6 in the presence of LPS. Our results suggest that BJcuL can modulate macrophage functional activation towards an M1 state.

Phagocytic activity of LPS tolerant macrophages

Endotoxin tolerance is defined as a reduced capacity of the host to respond to LPS activation following a first exposure to this stimulus. It affects all leukocytes and regarding macrophages, most studies focus on the reduced ability of these cells to secrete pro-inflammatory cytokines. Therefore, we evaluated other macrophages functions (fungicidal capacity, reactive oxygen species production and antigen presentation) in cells from tolerant mice. We have performed a tolerance model in our laboratory that does not stimulate directly the place from where the cells will be removed (peritoneal cavity). Mouse received subcutaneous injections of LPS in the scruff for 5 days and we analyze the capacity of peritoneal macrophages to phagocyte using three different receptors: Fc, C3b and mannose receptors. We found a reduction in the phagocytosis of erythrocytes and Candida albicans related to the Fc and mannose receptors. These differences can be due to a macrophage reprogramming, as demonstrated by altered expression of cytokines and chemokines. Despite this reduction in phagocytosis capacity, macrophages from tolerant animals exhibited enhanced hydrogen peroxide production and expression of antigen presentation molecules, suggesting that their ability to combat an infection is improved. In summary, our data indicates that LPS tolerance drives macrophages from a predominant release of proinflammatory mediators that amplify inflammation and host damage toward a better killing and antigen presentation state.

Association of high T-cell immune infiltrate and low hemorrhage in melanoma brain metastases (MBMs) with prolonged survival.

8528 Background: Despite the poor prognosis of patients (pts) with MBM several pts have prolonged survival. We hypothesized that the heterogeneity of BrMM is determined by differences in melanoma biology and its brain microenvironment. Methods: We identified pts who have undergone craniotomy for MBMs. Evaluation entailed complete clinical information, acquisition of archived melanoma brain metastases, histopathologic analysis of hematoxylin and eosin-stained sections (n=101), whole genome expression profiling (WGEP, Illumina DASL) in 29 archived tissues. Results were validated by immunohistochemistry (IHC) or in situ hybridization (ISH). Results: In univariate analysis (log-rank) factors significantly associated with prolonged survival were high immune infiltrate (HII) plus low hemorrhage (hazard ratio, HR, 2.71, p<0.001), present melanin (1.67, p=0.03), and recursive partitioning analysis (RPA) class 1 (0.37, p<0.0001). No association between HII and use of immunotherapy prior to craniotomy was noted. Only RPA class 1 (p=0.029) and HII plus low hemorrhage (p=0.002) remained significant in Cox proportional hazards model analysis. Gene set analysis of WGEP data confirmed that Encarta pathways related with T-cell activation and differentiation were significantly associated with prolonged survival whereas Y branching of actin filaments, presenilin action in Notch and Wnt signaling, G-protein signaling through tubby protein, lissencephaly gene in neuronal migration and development are among the gene categories associated with worse survival. IHC and ISH analysis of tumor sections for various markers (n=40) showed that high peritumoral CD3+ (3.31, p=0.009), high peritumoral CD4+ (4.41, p=0.014), and high peritumoral CD8+ (3.02, p=0.030) are associated with prolonged survival whereas neither CD14+ nor FoxP3+ infiltrate, nor high melanoma expression of antigen presentation molecules are associated with survival. Conclusions: Our study is the first to document that high peritumoral T-cell infiltrates are associated with prolonged survival. High tumor hemorrhage, an adverse prognostic sign, reflects aggressive melanoma cells that migrate and invade in the brain.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naïve subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and FcγRI (CD64) receptor ( P ≤ 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells ( P = 0.0356 and P = 0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-α, IL-12p40 and IFN-γ ( P ≤ 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection.

Enhancement of antigen-presenting cell surface molecules involved in cognate interactions by immunostimulatory DNA sequences.

Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN) containing immunostimulatory DNA sequences (ISS) have been proposed to foster a Th1 response via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1, B7-2 and CD40), cytokine receptors (IFN-gamma receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an Fc receptor (Fcgamma receptor) on murine B cells or bone marrow-derived macrophages. The increased expression of these surface molecules is seen in purified cell populations and is largely independent of the effects of type 1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their differentiation toward a Th1 phenotype in vitro. Thus, the induction of both type 1 cytokines and a distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity.