In Silico Tools for Predicting Novel Epitopes.

Identifying antigens within a pathogen is a critical task to develop effective vaccines and diagnostic methods, as well as understanding the evolution and adaptation to host immune responses. Historically, antigenicity was studied with experiments that evaluate the immune response against selected fragments of pathogens. Using this approach, the scientific community has gathered abundant information regarding which pathogenic fragments are immunogenic. The systematic collection of this data has enabled unraveling many of the fundamental rules underlying the properties defining epitopes and immunogenicity, and has resulted in the creation of a large panel of immunologically relevant predictive (in silico) tools. The development and application of such tools have proven to accelerate the identification of novel epitopes within biomedical applications reducing experimental costs. This chapter introduces some basic concepts about MHC presentation, T cell and B cell epitopes, the experimental efforts to determine those, and focuses on state-of-the-art methods for epitope prediction, highlighting their strengths and limitations, and catering instructions for their rational use.

The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.

Poxvirus Immune Evasion.

Poxviruses have evolved a wide array of mechanisms to evade the immune response, and we provide an overview of the different immunomodulatory strategies. Poxviruses prevent the recognition of viral DNA that triggers the immune responses and inhibit signaling pathways within the infected cell. A unique feature of poxviruses is the production of secreted proteins that mimic cytokines and cytokine receptors, acting as decoy receptors to neutralize the activity of cytokines and chemokines. The capacity of these proteins to evade cellular immune responses by inhibiting cytokine activation is complemented by poxviruses' strategies to block natural killer cells and cytotoxic T cells, often through interfering with antigen presentation pathways. Mechanisms that target complement activation are also encoded by poxviruses. Virus-encoded proteins that target immune molecules and pathways play a major role in immune modulation, and their contribution to viral pathogenesis, facilitating virus replication or preventing immunopathology, is discussed.

EGC enhances tumor antigen presentation and CD8+ T cell-mediated antitumor immunity via targeting oncoprotein SND1.

The Staphylococcal nuclease and Tudor domain containing 1 (SND1) has been identified as an oncoprotein. Our previous study demonstrated that SND1 impedes the major histocompatibility complex class I (MHC-I) assembly by hijacking the nascent heavy chain of MHC-I to endoplasmic reticulum-associated degradation. Herein, we aimed to identify inhibitors to block SND1-MHC-I binding, to facilitate the MHC-I presentation and tumor immunotherapy. Our findings validated the importance of the K490-containing sites in SND1-MHC-I complex. Through structure-based virtual screening and docking analysis, (-)-Epigallocatechin (EGC) exhibited the highest docking score to prevent the binding of MHC-I to SND1 by altering the spatial conformation of SND1. Additionally, EGC treatment resulted in increased expression levels of membrane-presented MHC-I in tumor cells. The C57BL/6J murine orthotopic melanoma model validated that EGC increases infiltration and activity of CD8+ T cells in both the tumor and spleen. Furthermore, the combination of EGC with programmed death-1 (PD-1) antibody demonstrated a superior antitumor effect. In summary, we identified EGC as a novel inhibitor of SND1-MHC-I interaction, prompting MHC-I presentation to improve CD8+ T cell response within the tumor microenvironment. This discovery presents a promising immunotherapeutic candidate for tumors.

Lactate Dehydrogenase-Elevating Virus Infection Inhibits MOG Peptide Presentation by CD11b+CD11c+ Dendritic Cells in a Mouse Model of Multiple Sclerosis.

Infections may affect the course of autoimmune inflammatory diseases of the central nervous system (CNS), such as multiple sclerosis (MS). Infections with lactate dehydrogenase-elevating virus (LDV) protected mice from developing experimental autoimmune encephalomyelitis (EAE), a mouse counterpart of MS. Uninfected C57BL/6 mice immunized with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) experienced paralysis and lost weight at a greater rate than mice who had previously been infected with LDV. LDV infection decreased the presentation of the MOG peptide by CD11b+CD11c+ dendritic cells (DC) to pathogenic T lymphocytes. When comparing non-infected mice to infected mice, the histopathological examination of the CNS showed more areas of demyelination and CD45+ and CD3+, but not Iba1+ cell infiltration. These results suggest that the protective effect of LDV infection against EAE development is mediated by a suppression of myelin antigen presentation by a specific DC subset to autoreactive T lymphocytes. Such a mechanism might contribute to the general suppressive effect of infections on autoimmune diseases known as the hygiene hypothesis.

CD8+ T cell targeting of tumor antigens presented by HLA-E.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.

Dendritic cells in Parkinson's disease: Regulatory role and therapeutic potential.

Parkinson's Disease (PD) is a debilitating neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons and the presence of Lewy bodies. While the traditional focus has been on neuronal and glial cell dysfunction, recent research has shifted towards understanding the role of the immune system, particularly dendritic cells (DCs), in PD pathogenesis. As pivotal antigen-presenting cells, DCs are traditionally recognized for initiating and regulating immune responses. In PD, DCs contribute to disease progression through the presentation of α-synuclein to T cells, leading to an adaptive immune response against neuronal elements. This review explores the emerging role of DCs in PD, highlighting their potential involvement in antigen presentation and T cell immune response modulation. Understanding the multifaceted functions of DCs could reveal novel insights into PD pathogenesis and open new avenues for therapeutic strategies, potentially altering the course of this devastating disease.

Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

Conventional type 1 dendritic cells (cDC1) are the essential antigen-presenting DC subset in antitumor immunity. Suppressing B-cell lymphoma 9 and B-cell lymphoma 9-like (BCL9/BCL9L) inhibits tumor growth and boosts immune responses against cancer. However, whether oncogenic BCL9/BCL9L impairs antigen presentation in tumors is still not completely understood. Here, we show that targeting BCL9/BCL9L enhanced antigen presentation by stimulating cDC1 activation and infiltration into tumor. Pharmacological inhibition of BCL9/BCL9L with a novel inhibitor hsBCL9z96 or Bcl9/Bcl9l knockout mice markedly delayed tumor growth and promoted antitumor CD8+ T cell responses. Mechanistically, targeting BCL9/BCL9L promoted antigen presentation in tumors. This is due to the increase of cDC1 activation and tumor infiltration by the XCL1-XCR1 axis. Importantly, using single-cell transcriptomics analysis, we found that Bcl9/Bcl9l deficient cDC1 were superior to wild-type (WT) cDC1 at activation and antigen presentation via NF-κB/IRF1 signaling. Together, we demonstrate that targeting BCL9/BCL9L plays a crucial role in cDC1-modulated antigen presentation of tumor-derived antigens, as well as CD8+ T cell activation and tumor infiltration. Targeting BCL9/BCL9L to regulate cDC1 function and directly orchestrate a positive feedback loop necessary for optimal antitumor immunity could serve as a potential strategy to counter immune suppression and enhance cancer immunotherapy.

Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.

Podocyte SIRPα reduction aggravates lupus nephritis via promoting T cell inflammatory responses.

Signal-regulatory protein alpha (SIRPα) has recently been found to be highly expressed in podocytes and is essential for maintaining podocyte function. However, its immunoregulatory function in podocytes remains elusive. Here, we report that SIRPα controls podocyte antigen presentation in specific T cell activation via inhibiting spleen tyrosine kinase (Syk) phosphorylation. First, podocyte SIRPα under lupus nephritis (LN) conditions is strongly downregulated. Second, podocyte-specific deletion of SIRPα exacerbates renal disease progression in lupus-prone mice, as evidenced by an increase in T cell infiltration. Third, SIRPα deletion or knockdown enhances podocyte antigen presentation, which activates specific T cells, via enhancing Syk phosphorylation. Supporting this, Syk inhibitor GS-9973 prevents podocyte antigen presentation, resulting in a decrease of T cell activation and mitigation of renal disease caused by SIRPα knockdown or deletion. Our findings reveal an immunoregulatory role of SIRPα loss in promoting podocyte antigen presentation to activate specific T cell immune responses in LN.

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic ß-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic ß-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of ß-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.

In Situ Hydrogel Modulates cDC1-Based Antigen Presentation and Cancer Stemness to Enhance Cancer Vaccine Efficiency.

Effective presentation of antigens by dendritic cells (DC) is essential for achieving a robust cytotoxic T lymphocytes (CTLs) response, in which cDC1 is the key DC subtype for high-performance activation of CTLs. However, low cDC1 proportion, complex process, and high cost severely hindered cDC1 generation and application. Herein, the study proposes an in situ cDC1 recruitment and activation strategy with simultaneous inhibiting cancer stemness for inducing robust CTL responses and enhancing the anti-tumor effect. Fms-like tyrosine kinase 3 ligand (FLT3L), Poly I:C, and Nap-CUM (NCUM), playing the role of cDC1 recruitment, cDC1 activation, inducing antigen release and decreasing tumor cell stemness, respectively, are co-encapsulated in an in situ hydrogel vaccine (FP/NCUM-Gel). FP/NCUM-Gel is gelated in situ after intra-tumoral injection. With the near-infrared irradiation, tumor cell immunogenic cell death occurred, tumor antigens and immunogenic signals are released in situ. cDC1 is recruited to tumor tissue and activated for antigen cross-presentation, followed by migrating to lymph nodes and activating CTLs. Furthermore, tumor cell stemness are inhibited by napabucasin, which can help CTLs to achieve comprehensive tumor killing. Collectively, the proposed strategy of cDC1 in situ recruitment and activation combined with stemness inhibition provides great immune response and anti-tumor potential, providing new ideas for clinical tumor vaccine design.

Polyclonally Derived Alloantigen-Specific T Regulatory Cells Exhibit Target-Specific Suppression and Capture MHC Class II from Dendritic Cells.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs in vitro and explored the fine specificity of their suppressive function and their mechanism of action in vitro and in vivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function in vivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

De novo identification of CD4+ T cell epitopes.

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.

TFEB regulates dendritic cell antigen presentation to modulate immune balance in asthma.

OBJECTIVE: Asthma stands as one of the most prevalent chronic respiratory conditions in children, with its pathogenesis tied to the actived antigen presentation by dendritic cells (DCs) and the imbalance within T cell subgroups. This study seeks to investigate the role of the transcription factor EB (TFEB) in modulating the antigen presentation process of DCs and its impact on the differentiation of T cell subgroups. METHODS: Bone marrow dendritic cells (BMDCs) were activated using house dust mites (HDM) and underwent RNA sequencing (RNA-seq) to pinpoint differentially expressed genes. TFEB mRNA expression levels were assessed in the peripheral blood mononuclear cells (PBMCs) of both healthy children and those diagnosed with asthma. In an asthma mouse model induced by HDM, the TFEB expression in lung tissue DCs was evaluated. Further experiments involved LV-shTFEB BMDCs co-cultured with T cells to explore the influence of TFEB on DCs' antigen presentation, T cell subset differentiation, and cytokine production. RESULTS: Transcriptomic sequencing identified TFEB as a significantly differentially expressed gene associated with immune system pathways and antigen presentation. Notably, TFEB expression showed a significant increase in the PBMCs of children diagnosed with asthma compared to healthy counterparts. Moreover, TFEB exhibited heightened expression in lung tissue DCs of HDM-induced asthmatic mice and HDM-stimulated BMDCs. Silencing TFEB resulted in the downregulation of MHC II, CD80, CD86, and CD40 on DCs. This action reinstated the equilibrium among Th1/Th2 and Th17/Treg cell subgroups, suppressed the expression of pro-inflammatory cytokines like IL-4, IL-5, IL-13, and IL-17, while augmenting the expression of the anti-inflammatory cytokine IL-10. CONCLUSION: TFEB might have a vital role in asthma's development by impacting the antigen presentation of DCs, regulating T cell subgroup differentiation, and influencing cytokine secretion. Its involvement could be pivotal in rebalancing the immune system in asthma. These research findings could potentially unveil novel therapeutic avenues for treating asthma.

Targeted acidosis mediated delivery of antigenic MHC-binding peptides.

Cytotoxic T lymphocytes are the primary effector immune cells responsible for protection against cancer, as they target peptide neoantigens presented through the major histocompatibility complex (MHC) on cancer cells, leading to cell death. Targeting peptide-MHC (pMHC) complex offers a promising strategy for immunotherapy due to their specificity and effectiveness against cancer. In this work, we exploit the acidic tumor micro-environment to selectively deliver antigenic peptides to cancer using pH(low) insertion peptides (pHLIP). We demonstrated the delivery of MHC binding peptides directly to the cytoplasm of melanoma cells resulted in the presentation of antigenic peptides on MHC, and activation of T cells. This work highlights the potential of pHLIP as a vehicle for the targeted delivery of antigenic peptides and its presentation via MHC-bound complexes on cancer cell surface for activation of T cells with implications for enhancing anti-cancer immunotherapy.

Tim4 enables large peritoneal macrophages to cross-present tumor antigens at early stages of tumorigenesis.

Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4+ large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.

Chaperone-mediated MHC-I peptide exchange in antigen presentation.

This work focuses on molecules that are encoded by the major histocompatibility complex (MHC) and that bind self-, foreign- or tumor-derived peptides and display these at the cell surface for recognition by receptors on T lymphocytes (T cell receptors, TCR) and natural killer (NK) cells. The past few decades have accumulated a vast knowledge base of the structures of MHC molecules and the complexes of MHC/TCR with specificity for many different peptides. In recent years, the structures of MHC-I molecules complexed with chaperones that assist in peptide loading have been revealed by X-ray crystallography and cryogenic electron microscopy. These structures have been further studied using mutagenesis, molecular dynamics and NMR approaches. This review summarizes the current structures and dynamic principles that govern peptide exchange as these relate to the process of antigen presentation.