Exogenous Antigens Research Articles

Computational analysis of peripheral blood RNA sequencing data unravels disrupted immune patterns in Alzheimer's disease.

The central nervous system (CNS) and the immune system collectively coordinate cellular functionalities, sharing common developmental mechanisms. Immunity-related molecules exert an influence on brain development, challenging the conventional view of the brain as immune-privileged. Chronic inflammation emerges as a key player in the pathophysiology of Alzheimer's disease (AD), with increased stress contributing to the disease progression and potentially exacerbating existing symptoms. In this study, the most significant gene signatures from selected RNA-sequencing (RNA-seq) data from AD patients and healthy individuals were obtained and a functional analysis and biological interpretation was conducted, including network and pathway enrichment analysis. Important evidence was reported, such as enrichment in immune system responses and antigen processes, as well as positive regulation of T-cell mediated cytotoxicity and endogenous and exogenous peptide antigen, thus indicating neuroinflammation and immune response participation in disease progression. These findings suggest a disturbance in the immune infiltration of the peripheral immune environment, providing new challenges to explore key biological processes from a molecular perspective that strongly participate in AD development.

MicroRNA transcriptome analysis reveals the immune regulatory mechanism of Crassostrea hongkongesis against Vibrio harveyi infection

MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate target-genes expression and play crucial roles in post-transcriptional regulation and immune system regulation. The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. Vibrio harveyi, a Gram negative luminescent marine bacterium, is widely distributed in coastal water environments and can cause large-scale death of C. hongkongesis. However, little in formation is available on the immune regulatory mechanisms of C. hongkongesis infected with V. harveyi. Therefore, we performed microRNA transcriptome analysis for elucidating the immunoregulation mechanism of C. hongkongesis infected with V. harveyi. The results show that a total of 308468208 clean reads and 288371159 clean tags were obtained. 222 differentially expressed miRNAs were identified. A total of 388 target genes that were differentially expressed and negatively correlated with miRNA expression were predicted by 222 DEmiRs. GO enrichment analysis of 388 DETGs showed that they were mainly enriched in the immune-related term of membrane-bounded vesicle, endocytic vesicle lumen, antigen processing and presentation of exogenous peptide antigen via MHC class I, antigen processing and presentation of peptide antigen via MHC class I, and other immune-related term. KEGG enrichment analysis showed that DETGs were mainly enriched in the Complement and coagulation cascades, Herpes simplex virus 1 infection, Bacterial invasion of epithelial cells, Antigen processing and presentation and NOD-like receptor signaling pathway. The 16 key DEmiRs and their target genes form a regulatory network for seven immune-related pathways. These results suggest that V. harveyi infection induces a complex miRNA response with wide-ranging effects on immune gene expression in the C. hongkongesis. This study explored the immune response of C. hongkongesis to V. harveyi infection at the level of miRNAs, which provides new ideas for the healthy culture and selective breeding of C. hongkongesis.

The fate of immune complexes in membranous nephropathy.

The most characteristic feature of membranous nephropathy (MN) is the presence of subepithelial electron dense deposits and the consequential thickening of the glomerular basement membrane. There have been great advances in the understanding of the destiny of immune complexes in MN by the benefit of experimental models represented by Heymann nephritis. Subepithelial immune complexes are formed in situ by autoantibodies targeting native autoantigens or exogenous planted antigens such as the phospholipase A2 receptor (PLA2R) and cationic BSA respectively. The nascent immune complexes would not be pathogenic until they develop into immune deposits. Podocytes are the major source of autoantigens in idiopathic membranous nephropathy. They also participate in the modulation and removal of the immune complexes to a large extent. The balance between deposition and clearance is regulated by a wide range of factors such as the composition and physicochemical properties of the immune complexes and the complement system. Complement components such as C3 and C1q have been reported to be precipitated with the deposits whereas a complement regulatory protein CR1 expressed by podocytes is involved in the phagocytosis of immune complexes by podocytes. Podocytes regulate the dynamic change of immune complexes which is disturbed in membranous nephropathy. To elucidate the precise fate of the immune complexes is essential for developing more rational and novel therapies for membranous nephropathy.

Molecular Characteristics, Functional Definitions, and Regulatory Mechanisms for Cross-Presentation Mediated by the Major Histocompatibility Complex: A Comprehensive Review.

The major histocompatibility complexes of vertebrates play a key role in the immune response. Antigen-presenting cells are loaded on MHC I molecules, which mainly present endogenous antigens; when MHC I presents exogenous antigens, this is called cross-presentation. The discovery of cross-presentation provides an important theoretical basis for the study of exogenous antigens. Cross-presentation is a complex process in which MHC I molecules present antigens to the cell surface to activate CD8+ T lymphocytes. The process of cross-representation includes many components, and this article briefly outlines the origins and development of MHC molecules, gene structures, functions, and their classical presentation pathways. The cross-presentation pathways of MHC I molecules, the cell lines that support cross-presentation, and the mechanisms of MHC I molecular transporting are all reviewed. After more than 40 years of research, the specific mechanism of cross-presentation is still unclear. In this paper, we summarize cross-presentation and anticipate the research and development prospects for cross-presentation.

An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region– positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Regulation of Macrophage Polarization in Allergy by Noncoding RNAs.

Allergy is a type 2 immune reaction triggered by antigens known as allergens, including food and environmental substances such as peanuts, plant pollen, fungal spores, and the feces and debris of mites and insects. Macrophages are myeloid immune cells with phagocytic abilities that process exogenous and endogenous antigens. Upon activation, they can produce effector molecules such as cytokines as well as anti-inflammatory molecules. The dysregulation of macrophage function can lead to excessive type 1 inflammation as well as type 2 inflammation, which includes allergic reactions. Thus, it is important to better understand how macrophages are regulated in the pathogenesis of allergies. Emerging evidence highlights the role of noncoding RNAs (ncRNAs) in macrophage polarization, which in turn can modify the pathogenesis of various immune-mediated diseases, including allergies. This review summarizes the current knowledge regarding this topic and considers three classes of ncRNAs: microRNAs, long ncRNAs, and circular ncRNAs. Understanding the roles of these ncRNAs in macrophage polarization will provide new insights into the pathogenesis of allergies and identify potential novel therapeutic targets.

Aplastic Anemia Patients Have Diverse T Cell Repertoires and Flexible T Cell Responses Similar to Healthy Individuals

Acquired aplastic anemia (AA) is a bone marrow failure disease caused by T cell-mediated autoimmune attack on hematopoietic stem and progenitor cells (HSPCs). Identifying autoreactive T cells in AA has been challenging. Advances in T cell receptor (TCR) sequencing and the discovery of Human Leukocyte Antigen Class I (HLA-I) risk alleles linked to autoantigen presentation in AA have rekindled interest in exploring T cell repertoires in AA. We hypothesized that autoreactive CD8 + T cells in AA may have convergent TCR structures allowing them to recognize AA autoantigens presented by specific HLA-I risk alleles. To test our hypothesis, we analyzed TCR repertoires and HLA-restricted CD8 + T cell reactivity in AA patients. Using TCR Vβ (TRB) sequencing, we analyzed 83 patient samples obtained at AA diagnosis or relapse. High-risk alleles HLA-B*14:02 and B*40:02 were present in 26% and 10% of patients, respectively. HLA-DR*15 was present in 24%. Patient samples were largely sorted bone marrow (BM) CD8 + T lymphocytes. Patient TRB repertoires were compared to those in 709 control samples, largely of unsorted peripheral blood (PB). To account for sampling differences, we downsampled sequences to the minimum number of clones in the matching tissue. Normalized clone size distributions were visualized on rank plots (Fig 1). TRB repertoires in both patients and controls were diverse. Highly expanded clones (>5%) were rare in either group. Patients had 1,415,069 unique CDR3 amino acid sequences (CDR3AA), of which 106,446 (7.5%) were shared by ≥2 patients. Among the 106,446 shared CDR3AA, 95.4% were found in controls, and 4.3% TCRs had reported specificity to exogenous (mostly viral) or endogenous antigens in VDJdb. 4,920 (4.6%) of the shared CDR3AA were detected only in patients (median 2 patients (2-25)). Of these, 190 exhibited TCR convergence in ≥1 patient, such that the same CDR3AA was encoded by ≥2 distinct nucleotide sequences. Clustering 4,920 patient-associated CDR3AA using GLIPH (Grouping of lymphocyte interactions by paratope hotspots) identified 43 TCR antigen convergence groups. CDR3AA in each cluster were not significantly expanded in the majority of patients and were not enriched for the high-risk alleles HLA-B*14:02 or B*40:02. Our data suggest that most CDR3AA in AA are private (unique to individual patients). Shared autoreactive CDR3AA may exist in patients but are likely to also be present in healthy individuals. As the next step towards identifying autoreactive T cells, we focused on AA patients with known HLA-I risk alleles. We tested their CD8 + T cells for reactivity with endogenously expressed antigens by stimulating T cells with hematopoietic cell lines engineered to express single HLA-I alleles matching those of the patients (Fig 2). TRB sequencing showed increased clonality and significant expansion of a subset of clonotypes after T cell stimulation. Interferon γ enzyme-linked immunospot assays confirmed the expanded T cells' responsiveness to antigens presented by HLA alleles used for T cell stimulation. Additionally, these expanded T cells demonstrated variable reactivity to cells expressing other HLA alleles from the same patient. Among the 1% of CDR3AA clonotypes most highly expanded with single HLA-I alleles (63-3000-fold), we identified several that were expanded in an HLA-restricted manner. However, several highly expanded CDR3AA were highly expanded with multiple HLA alleles. Potential reasons for expansion of some CDR3AA after stimulation with cells expressing different HLA alleles include 1) binding of the same CDR3AA to different antigens presented by distinct HLA alleles, 2) expansion in response to the same antigens presented by distinct HLA alleles, or 3) expansion in response to non-HLA antigens. In conclusion, our data show diverse and mostly private TRB repertoires in AA patients. Our in vitro findings suggest that much like T cell responses in non-AA individuals, T cell responses in AA are diverse and exhibit flexibility with respect to activation in response to various endogenous antigens in the context of different self HLA-I alleles. Given the diversity of TCR responses observed in AA patients, the tissue specificity of autoimmune attack and established association with risk HLA-I alleles in AA remain a mystery and likely require HSPC-specific autoantigen expression and efficient autoantigen presentation by individual patients' HLA-I alleles.

A Phase 2 Trial of SCRI-CAR19 Demonstrates Favorable Leukemia-Free Survival (LFS), with a Pilot Study of CD19-Expressing T-Cell Antigen Presenting Cells (CD19t T-APCs) Demonstrating Safety and Tolerability

The value of CD19 directed CAR T cell therapy for relapsed, refractory B-ALL has been well established. Iterative and complementary clinical trials have identified subgroups with improved outcomes, including those with low disease burden at the time of treatment as well as those with prolonged CAR T cell in vivo persistence. PLAT-02 was a phase 1/2 study of SCRI-CAR19, a 2 nd generation murine CAR with 41BB co-stimulation. Phase 1 previously reported 1 year LFS, event-free (EFS) and overall survival (OS) of 55%, 51.2%, and 69.5%. Subjects with low CD19 antigen burden in the bone marrow (<15%) prior to lymphodepletion (LD) and those with a rapid contraction of CAR T cells between Day 10 and 14 (ratio ≥1.5) were at higher risk of early loss of persistence. Phase 2 of PLAT-02 was a multisite trial along with a single site pilot study, PLAT-03, to explore administration of exogenous CD19 antigen designed to boost CAR T cell engraftment in subjects at high risk of early loss of persistence. PLAT-02 (NCT02028455) enrolled subjects 1-26 years of age with relapsed, refractory CD19+ B-ALL. The primary efficacy cohort was defined as those who received fludarabine/cyclophosphamide (flu/cy) LD and were infused with CAR19. PLAT-03 (NCT03186118) was a pilot study of CD19t T-APCs (manufactured T cells with CD19 tag) administered at doses of 10x10 6/kg or flat dosing of 5x10 8 for subjects >25kg every 4 weeks up to 6 total doses. Cohort A included subjects with low CD19 antigen burden, and cohort B included those with rapid early contraction of CAR19. 88 subjects with B-ALL were enrolled on PLAT-02 with plans for treatment at the recommended phase 2 dose (RP2D) of 1x10 6 CAR+ T cells/kg. Eighty-six percent had at least one prior relapse and 42% had undergone prior hematopoietic cell transplant (HCT). The feasibility of manufacturing was 98%. 71 subjects were in the primary efficacy cohort with an 89% minimal residual disease negative complete remission (MRDneg CR) rate. EFS and OS probability estimates with 95% CI at 1 year following CAR19 were 0.65 (0.53, 0.75) and 0.76 (0.64, 0.84). LFS at 1 year from entering MRDneg CR was 0.71 (0.58, 0.81). Cytokine release syndrome (CRS) was grade (Gr) 1 (53%), Gr 2 (26%), Gr 3 (10%), and Gr 4 (3%). Neurotoxicity occurred in 53%: Gr 1 (24%), Gr 2 (13%), Gr 3 (15%), and Gr 5 (1%). 25 subjects were treated on PLAT-03, 10 as cohort A (10 received CAR19 on PLAT-03, 9 received T-APCs), and 11 as cohort B (5 received CAR19 on PLAT-02, 6 received CAR19 on PLAT-03, and 9 total received T-APCs). One was cohort C (retreatment) and 3 were cohort D (no T-APCs). A total of 72 infusions of T-APCs were administered, with no occurrence of CRS or neurotoxicity. Two subjects had grade 3 toxicities related to T-APCs, 1 febrile neutropenia and 1 infusion related reaction. EFS and OS probability estimates at 1 year following CAR19 were 0.67 (0.43, 0.83) and 0.95 (0.71, 0.99). Overall LFS at 1 year was 0.63 (0.38, 0.80); by cohort: cohort A, 0.44 (0.13, 0.72) and cohort B, 0.80 (0.41, 0.95). Median duration of persistence, as determined by cumulative incidence using B cell aplasia (BCA) as a surrogate, among all subjects who received RP2D of CAR19 with flu/cy LD across PLAT-02 and PLAT-03, engrafted and achieved MRDneg CR, was 12.9 months (m). For subjects who did not receive T-APCs, the median persistence was 12.1m. Impact of antigen burden prior to LD was again observed; those with >15% CD19 antigen burden had persistence of >12.1m vs. 3.9m in the low antigen burden group. For subjects treated with T-APCs, the median persistence was 16m; 9.9m in cohort A and >16m in cohort B. The improvement in LFS after SCRI-CAR19 in phase 2 compared to phase 1 is likely multi-factorial, with contributing factors including earlier treatment, uniform use of LD, enhanced persistence, and better allocation of patients who benefit from consolidative HCT. The use of T-APCs was well tolerated without recurrent CAR T cell mediated toxicity. Although not directly comparable, there did appear to be enhancement of CAR T cell persistence with the use of T-APCs in populations predicted to have inferior persistence, including those with low antigen burden. There was not a clear improvement of LFS in those who received T-APCs. However, subjects were not randomized to receive T-APCs and those enrolled on PLAT-03 were more heavily treated and more likely to have received a prior HCT. Further exploration of T-APCs is warranted to define and evaluate their impact on persistence and durable responses.

Evidence of Defective Cell-Autonomous Signalling By B Cell Receptors in CLL Stereotyped Subset #1

B cell receptor (BCR)-mediated signalling is central in the pathogenesis of chronic lymphocytic leukaemia (CLL), and represents a target for pharmacological intervention. Notably, so far characterized CLL-derived BCRs are capable of triggering cell-autonomous intracellular signalling through homotypic interactions without requirement for exogenous antigens. Approximately 41% of all CLL cases can be classified in subsets based on homologous, otherwise known as stereotyped, BCRs expressed by the malignant clones, sharing remarkably similar sequences in the VH CDR3 region and highly restricted immunoglobulin heavy and/or light variable genes. Since cases within a subset display clinicobiological consistency in contrast with the pronounced heterogeneity of CLL at large, analysis of the mechanism of BCR-mediated signalling can unveil correlations between molecular details of BCR activation and the course of the disease. Our present study focused on BCRs of stereotyped subset #1, the second largest stereotyped subset, known for a particularly aggressive clinical course. Cases belonging to subset #1 express unmutated IGHV genes of phylogenetic clan I (namely, IGHV1, IGHV5 and IGHV7 subgroup genes) partnered by light chains encoded by the IGKV1-39/1D-39 gene. First, we used X-ray crystallography to determine the high-resolution crystal structures of the BCR Fab fragments from three subset #1 cases utilizing the IGHV1-2, IGHV1-3, and IGHV5-10 genes partnered with IGKV1-39/1D-39 to obtain information on the combining sites' structures and possible modes of intermolecular interactions that may mediate cell-autonomous signalling. Analysis of the experimental structures revealed that, despite the high sequence homology of the three proteins, the heavy and light chain CDR loops in the three BCR Fabs are arranged differently to shape binding cavities with distinct dimensions and polarity, thus suggesting that distinct antigens may be the physiological target of each subgroup. Unlike BCRs from other subsets previously characterised, the intermolecular contacts between the subset #1 Fab molecules in the three crystals involve diverse residues that are not indicative of a shared homotypic interaction. In addition, the surfaces involved in the contacts are small and the computed energies for the protein-protein contacts observed are suggestive at best of very weak interactions. In accordance to this, when we used analytical ultracentrifugation along with chromatographic and calorimetric methods to detect BCR Fab oligomerization in solution, no evidence of self-association was obtained, thus indicating that the receptors in solution either do not interact, or are characterized by self-dissociation constants that exceed the sensitivity limit of the techniques employed. To assess whether the cellular environment could promote self-association and signalling activity of the full-length BCR, we expressed subset #1 BCRs in the model triple knockout (TKO) pre-B-cell line, designed to measure Ca 2+ ion influx using cytofluorimetry. All subset #1 BCRs tested were unable to mobilize autonomously the Ca 2+ ions, despite being fully expressed on the cell surface and capable of signalling upon stimulation with anti-BCR antibody as a proxy for exogenous ligand. This result is in sharp contrast with the behaviour of all other CLL-derived BCRs so far characterised in this system that could activate a cell-autonomous signal as evidenced from a persistent Ca 2+ ion influx in the transduced cells without requiring external stimuli. Hence, subset #1 BCRs differ from the so far characterized BCRs from CLL cases belonging to other stereotyped subsets, including clinically aggressive subset #2 and its satellite subset #169 as well as the clinically indolent stereotyped subset #4. In conclusion, our combined biochemical and cellular analysis demonstrates that a constitutively active cell-autonomous signalling may not be a unifying feature of all CLL clones as previously proposed, and that other mechanisms may be at the roots of the BCR-mediated signal required for leukemic cell proliferation and development of CLL.

Expression and Immune Characterization of Major Histocompatibility Complex in Paralichthys olivaceus after Antigen Stimulation.

The Major histocompatibility complex (Mhc) is an important molecule for antigen presenting and binds to T cell receptors, activating T lymphocytes and triggering specific immune responses. To investigate the role of MhcII in adaptive immunity, in this study, mhcIIα and mhcIIβ of flounder (Paralichthys olivaceus) were cloned, polyclonal antibodies (Abs) against their extracellular regions were produced, respectively, and their distribution on cells and tissues and expression patterns, which varied by antigen stimulation or pathogen infection, were investigated. The results showed that the open reading frame (ORF) of mhcIIα is 708 bp, including 235 amino acids (aa); and the ORF of mhcIIβ is 741 bp, encoding 246aa. The mhcIIα and mhcIIβ were significantly expressed in gills, spleen, and peripheral blood leukocytes (PBLs). Their antibodies could specifically recognize eukaryotic expressed MhcIIα and MhcIIβ. MhcIIα+ and MhcIIβ+ cells were 30.2 ± 2.9% of the percentage in peripheral blood leukocytes. MhcII molecules were co-localized with CD83 and IgM on leukocytes, respectively, but not on CD4+ or CD8+ T lymphocyte subpopulations. The expression of both mhcIIα and mhcIIβ were significantly upregulated in flounder after bacteria and virus challenges. The percentages of MhcII+ cells, MhcII+/CD83+, and MhcII+/IgM+ double-positive cells increased significantly after PHA and ConA stimulation, respectively; they varied significantly in PBLs after polyI:C stimulation, and no variations were found after LPS treatment. In the meantime, variations in MhcII+ cells were consistent with that of CD4+ T lymphocytes. These results suggest that MhcII, mainly expressed in B cells and dendritic cells, play an essential role in antigen presentation, and respond significantly to exogenous antigens and T cell-dependent antigens. These results may provide an important reference for the study of cellular immunity in teleosts.

Increases in Cellular Immune Responses Due to Positive Effect of CVC1302-Induced Lysosomal Escape in Mice.

This study found a higher percentage of CD8+ T cells in piglets immunized with a CVC1302-adjuvanted inactivated foot-and-mouth disease virus (FMDV) vaccine. We wondered whether the CVC1302-adjuvanted inactivated FMDV vaccine promoted cellular immunity by promoting the antigen cross-presentation efficiency of ovalbumin (OVA) through dendritic cells (DCs), mainly via cytosolic pathways. This was demonstrated by the enhanced levels of lysosomal escape of OVA in the DCs loaded with OVA and CVC1302. The higher levels of ROS and significantly enhanced elevated lysosomal pH levels in the DCs facilitated the lysosomal escape of OVA. Significantly enhanced CTL activity levels was observed in the mice immunized with OVA-CVC1302. Overall, CVC1302 increased the cross-presentation of exogenous antigens and the cross-priming of CD8+ T cells by alkalizing the lysosomal pH and facilitating the lysosomal escape of antigens. These studies shed new light on the development of immunopotentiators to improve cellular immunity induced by vaccines.

The inflammatory response to birth requires MyD88 and is driven by both mother and offspring

Birth is an inflammatory event for the newborn, characterized by elevations in interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α peripherally and/or centrally, as well as changes in brain microglia. However, the mechanism(s) underlying these responses is unknown. Toll-like receptors (TLRs) play crucial roles in innate immunity and initiate inflammatory cascades upon recognition of endogenous or exogenous antigens. Most TLR signaling depends on the adaptor molecule myeloid differentiation primary response 88 (MyD88). We independently varied MyD88 gene status in mouse dams and their offspring to determine whether the inflammatory response to birth depends on MyD88 signaling and, if so, whether that signaling occurs in the offspring, the mother, or both. We find that the perinatal surges in plasma IL-6 and brain expression of TNF-α depend solely on MyD88 gene status of the offspring, whereas postnatal increases in plasma IL-10 and TNF-α depend on MyD88 in both the pup and dam. Interestingly, MyD88 genotype of the dam primarily drives differences in offspring brain microglial density and has robust effects on developmental neuronal cell death. Milk cytokines were evaluated as a possible source of postnatal maternal influence; although we found high levels of CXCL1/GROα and several other cytokines in ingested post-partum milk, their presence did not require MyD88. Thus, the inflammatory response previously described in the late-term fetus and newborn depends on MyD88 (and, by extension, TLRs), with signaling in both the dam and offspring contributing. Unexpectedly, naturally-occuring neuronal cell death in the newborn is modulated primarily by maternal MyD88 gene status.

EXTH-02. A TERT-SPECIFIC TCR-LIKE ANTIBODY CAR T CELL APPROACH FACILITATES KILLING OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a difficult form of brain cancer to treat as GBM cells utilize various strategies to evade host immune responses. Prior studies have shown autologous cytotoxic T cells (CTL) modified to express chimeric antigen receptors (CAR) facilitating host CTL responses against cancers. This study investigates the ability of T-cell receptor (TCR)-like antibody clones against TERT to facilitate specific CAR T cell mediated killing of GBM. Lentiviral vectors were created for five TCR-like antibody CAR constructs recognizing an HLA-A*02:01-restricted TERT-derived antigen. Nur77-GFP Jurkat reporter cell lines and 3 healthy donor-derived CTLs transduced with these vectors were used in flow-based and in vitro killing assays to assess specificity and functionality. K562 cells that did, and did not, express HLA-A*02:01 were targets in the flow-based assay. Flow cytometry captured activation of transduced Jurkat cells via GFP and CD69 upregulation. Three luciferase-transduced GBM cell lines were targets in the cytotoxic killing assay and visualized via bioluminescence imaging (BLI). Increased GFP and CD69 expression was seen on Jurkat cells transduced with clones 3A12 and 3H2 only when co-cultured with TERT antigen and A*02 positive K562 suggesting specific recognition of the TERT:HLA complex. Furthermore, healthy donor-derived CTL transduced with 3A12 and 3H2 CAR constructs demonstrated in vitro killing of tumor cells by BLI. Interestingly, the 3H2 construct only showed cytotoxicity in the presence of exogenous TERT antigen. However, when the heavy and light chains of the 3H2 clone were flipped in the lentiviral construct (3H2F), specificity to the TERT:HLA complex was preserved but the CAR T cells now demonstrated cytotoxicity of GBM without needing exogeneous peptide. TCR-like antibody clones 3A12 and 3H2F specifically recognize an HLA-A*02:01-restricted TERT-derived antigen, facilitating effective in vitro cytotoxicity of GBM cells. Ongoing experiments will evaluate the in vivo activity of these TERT-specific CAR constructs and assess potential off-target toxicity.

STEM-04. FATE-BASED CONVERSION FOR IN SITU VACCINATION IN GLIOBLASTOMA

Abstract BACKGROUND Although immune checkpoint inhibitors have shown high benefit for other solid tumors, their efficacy is limited in GBM due in large part to the “cold” tumor microenvironment (TME) of the GBM tumor. New approaches are necessary to overcome these challenges. We propose a shift in the scientific and treatment paradigm of cancer immunotherapy, calling for tumor cells to take on a central, active role in initiating their own immunity right in the TME. METHODS Using AI, we identified cell fate determinants (CFDs) to convert GBM cells directly into induced dendritic cells (iDC) inside the TME. CFDs were delivered using a viral vector and conversion assessed by immunophenotyping, scRNA-seq, and functional assays for classical DC properties. RESULTS A four-CFD subnetwork anchored by PU.1 was sufficient to convert mouse GBM cells to iDCs (CD45+MHCII+CD80/86+MHCI+). iDCs are growth arrested, exhibit 3-fold higher phagocytic activity and upregulate the canonical antigen processing and presenting machineries by 10-40-fold, resulting in 100-fold greater efficiency at processing ovalbumin and cross-presenting SIINFEKL to naive OTI-CD8+ cytotoxic T lymphocytes (CTL). In addition, iDCs efficiently cross-present exogenous antigens to naïve CD8+ CTLs and elicit >30-fold higher activation and cytotoxicity in tumor-specific T cells compared to native GBM cells, confirming their DC-like properties. Lastly, intratumoral GBM-DC conversion in a syngeneic orthotopic GBM model resulted in significant reduction in tumor burden and increase in survival compared to controls (Log-rank, p = 0.01) with synergistic efficacy with ICIs and classical DC-based tumor vaccination. We have also successfully converted human GBM cells to iDCs with similar properties using a 2-CFD combination also anchored by PU.1. CONCLUSIONS GBM-derived iDCs acquired DC-like functions and were capable of eliciting robust intratumor immune activation, thereby overcoming many current limitations of immunotherapy in GBM. Our goal is to develop a low cost, off-the-shelf gene therapy-based fate conversion tumor immunotherapy.

Liposome Vaccine for Active Regulation of Cellular and Humoral Immunity.

Despite significant progress in vaccine development, especially in the fight against viral infections, many unexplored areas remain including innovative adjuvants, diversification of vaccine formulations, and research into the coordination of humoral and cellular immune mechanisms induced by vaccines. Effective coordination of humoral and cellular immunity is crucial in vaccine design. In this study, we used the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or ovalbumin (OVA) as antigen models and CpG DNA (an activator of toll-like receptor 9, TLR9) as an adjuvant to prepare a multitargeted liposome (LIPO) vaccine. Once equipped with the ability to target lymph nodes (LN) and the endoplasmic reticulum (ER), the LIPO vaccine significantly enhances the cross-presentation ability of antigen-presenting cells (APCs) for exogenous antigens through the ER-associated protein degradation (ERSD) mechanism. Additionally, the vaccine could fine-tune the efficiency of ER-targeted antigen delivery, actively regulating the presentation of exogenous antigen proteins via the major histocompatibility complex (MHC-I) or MHC-II pathways. Immune data from in vivo mouse experiments indicated that the LIPO vaccine effectively stimulated both humoral and cellular immune responses. Furthermore, it triggers immune protection by establishing a robust and persistent germinal center. Moreover, the multifunctionality of this LIPO vaccine extends to the fields of cancer, viruses, and bacteria, providing insights for skilled vaccine design and improvement.

Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity.

Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

Major histocompatibility complex class I assembly within endolysosomal pathways.

Major histocompatibility complex classI (MHC classI) molecules facilitate subcellular immune surveillance by presenting peptides on the cell surface. MHC classI assembly with peptides generally happens in the endoplasmic reticulum (ER). Peptides are processed in the cytosol, transported into the ER, and assembled with MHC classI heavy and light chains. However, as many pathogens reside within multiple subcellular organelles, peptide sampling across non-cytosolic compartments is also important. MHC classI molecules internalize from the cell surface into endosomes and constitutively traffic between endosomes and the cell surface. Within endosomes, MHC classI molecules assemble with both exogenous and endogenous antigens processed within these compartments. Human MHC classI polymorphisms, well known to affect ER assembly modes, also influence endosomal assembly outcomes, an area of current interest to the field.

Controlling Antigen Fate in Therapeutic Cancer Vaccines by Targeting Dendritic Cell Receptors.

Antigen-presenting cells (APCs) orchestrate immune responses and are therefore of interest for the targeted delivery of therapeutic vaccines. Dendritic cells (DCs) are professional APCs that excel in presentation of exogenous antigens toward CD4+ T helper cells, as well as cytotoxic CD8+ T cells. DCs are highly heterogeneous and can be divided into subpopulations that differ in abundance, function, and phenotype, such as differential expression of endocytic receptor molecules. It is firmly established that targeting antigens to DC receptors enhances the efficacy of therapeutic vaccines. While most studies emphasize the importance of targeting a specific DC subset, we argue that the differential intracellular routing downstream of the targeted receptors within the DC subset should also be considered. Here, we review the mouse and human receptors studied as target for therapeutic vaccines, focusing on antibody and ligand conjugates and how their targeting affects antigen presentation. We aim to delineate how targeting distinct receptors affects antigen presentation and vaccine efficacy, which will guide target selection for future therapeutic vaccine development.

Salmonella delivers H9N2 influenza virus antigens via a prokaryotic and eukaryotic dual-expression vector and elicits bivalent protection against avian influenza and fowl typhoid

The H9N2 avian influenza virus significantly affects the health of poultry and humans. We identified a prokaryotic and eukaryotic dual-expression vector system, pJHL270, that can provide simultaneous MHC class I and II stimulation of the host immune system, and we designed vaccine antigens by selecting the consensus HA1 sequence and M2e antigens from H9N2 virus circulating in South Korea from 2000 to 2021. The genes were cloned into the pJHL270 vector, and the cloned plasmid was delivered by a live-attenuated Salmonella Gallinarum (SG) strain. The immunity and protective efficacy of the SG-based H9N2 vaccine construct, JOL2922, against avian influenza and fowl typhoid (FT) were evaluated. The Ptrc and CMV promoters conferred antigen expression in prokaryotic and eukaryotic cells to induce balanced Th-1/Th-2 immunity. Chickens immunized with JOL2922 yielded high antigen-specific humoral and mucosal immune responses. qRT-PCR revealed that the strain generated polyfunctional IFN-γ and IL-4 secretion in immunized chickens. Furthermore, a FACS analysis showed increased CD3CD4+ and CD3CD8+ T-cell subpopulations following immunization. Peripheral Blood Mononuclear Cells (PBMCs) harvested from the immunized chickens significantly increased MHC class I and II expression, 3.5-fold and 2.5-fold increases, respectively. Serum collected from the immunized groups had an evident hemagglutinin inhibition titer of ≥6 log2. Immunization reduced the lung viral titer by 3.8-fold within 5 days post-infection. The strain also generated SG-specific humoral and cellular immune responses. The immunized birds all survived a virulent SG wild-type challenge. In addition, the bacterial burden was reduced by 2.7-fold and 2.1-fold in spleen and liver tissue, respectively, collected from immunized chickens. Our data indicate that an attenuated SG strain successfully delivered the dual-expression vector system and co-stimulated MHC class I and II antigen presentation pathways via exogenous and endogenous antigen presentation, thereby triggering a balanced Th-1/Th-2-based immune response and conferring effective protection against avian influenza and FT.

Histological and immunohistochemical characterization of mucosa-associated lymphoid tissue and antigen-presenting cells in trachea and lung of cattle.

The presence of bronchus-associated lymphoid tissue (BALT) and its structural components has been described in different healthy animal species and in animals with diseases of the respiratory tract. In contrast to normal mammals, BALT is absent in healthy human adult lungs, but has been found in the lungs of children. The histological characteristics of organized mucosa-associated lymphoid tissue (MALT), its subsets of immune cells and their in situ distribution in the lung of healthy subadult and adult cattle shows close similarities with BALT in humans and other animal species such as sheep, horses and pigs. This study clearly demonstrates that organized MALT also occurs in the tracheal mucosa of cattle. The absence of tracheal MALT and BALT in calves suggest that these structures are not constitutive. In the mucosa of bovine trachea, bronchi and bronchioli, MHC II+ and CD11c+ dendritic cells (DCs) are located in the epithelium and in the lamina propria mucosae. These DCs are already present in calves soon after birth. Examination of tangential epithelial sheets shows that in the bovine tracheal epithelium, like in man and rat, a dense network of MHC II+ and CD11c+ DCs exists and that their number is considerably higher than in conventional transverse sections. In the bovine tracheal and bronchial epithelium, MHC II+ DCs are extending their dendrites towards the lumen indicating that these DCs possibly are involved in sampling of luminal antigens. The presence of significantly higher numbers of MHC II+ DCs in the tracheal and bronchial/bronchiolar mucosa of older cattle in than in calves possibly results from local stimulation with exogenous antigens during postnatal life. Detection of DCs expressing the costimulatory molecules CD80 and CD86 in calves and cattle suggests maturation of DCs, which is most likely induced by stimulation with exogenous antigens.