Major Histocompatibility Complex Molecules Research Articles

Mechanisms of tumor immunosuppressive microenvironment formation in esophageal cancer.

As a highly invasive malignancy, esophageal cancer (EC) is a global health issue, and was the eighth most prevalent cancer and the sixth leading cause of cancer-related death worldwide in 2020. Due to its highly immunogenic nature, emer-ging immunotherapy approaches, such as immune checkpoint blockade, have demonstrated promising efficacy in treating EC; however, certain limitations and challenges still exist. In addition, tumors may exhibit primary or acquired resistance to immunotherapy in the tumor immune microenvironment (TIME); thus, understanding the TIME is urgent and crucial, especially given the im-portance of an immunosuppressive microenvironment in tumor progression. The aim of this review was to better elucidate the mechanisms of the suppressive TIME, including cell infiltration, immune cell subsets, cytokines and signaling pathways in the tumor microenvironment of EC patients, as well as the downregulated expression of major histocompatibility complex molecules in tumor cells, to obtain a better understanding of the differences in EC patient responses to immunotherapeutic strategies and accurately predict the efficacy of immunotherapies. Therefore, personalized treatments could be developed to maximize the advantages of immunotherapy.

Functional and biological implications of clonotypic diversity among human donor-unrestricted T cells.

Tcells express a T-cell receptor (TCR) heterodimer that is the product of germline rearrangement and junctional editing resulting in immense clonotypic diversity. The generation of diverse TCR repertoires enables the recognition of pathogen-derived peptide antigens presented by polymorphic major histocompatibility complex (MHC) molecules. However, Tcells also recognize nonpeptide antigens through nearly monomorphic antigen-presenting systems, such as cluster of differentiation 1 (CD1), MHC-related protein 1 (MR1) and butyrophilins (BTNs). This potential for shared immune responses across genetically diverse populations led to their designation as donor-unrestricted Tcells (DURTs). As might be expected, some CD1-, MR1- and BTN-restricted Tcells express a TCR that is conserved across unrelated individuals. However, several recent studies have reported unexpected diversity among DURT TCRs, and increasing evidence suggests that this diversity has functional consequences. Recent reports also challenge the dogma that immune cells are either innate or adaptive and suggest that DURT TCRs may act in both capacities. Here, we review this evidence and propose an expanded view of the role for clonotypic diversity among DURTs in humans, including new perspectives on how DURT TCRs may integrate their adaptive and innate immune functions.

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.

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.

Can AlphaFold's breakthrough in protein structure help decode the fundamental principles of adaptive cellular immunity?

T cells are essential immune cells responsible for identifying and eliminating pathogens. Through interactions between their T-cell antigen receptors (TCRs) and antigens presented by major histocompatibility complex molecules (MHCs) or MHC-like molecules, T cells discriminate foreign and self peptides. Determining the fundamental principles that govern these interactions has important implications in numerous medical contexts. However, reconstructing a map between T cells and their antagonist antigens remains an open challenge for the field of immunology, and success of in silico reconstructions of this relationship has remained incremental. In this Perspective, we discuss the role that new state-of-the-art deep-learning models for predicting protein structure may play in resolving some of the unanswered questions the field faces linking TCR and peptide-MHC properties to T-cell specificity. We provide a comprehensive overview of structural databases and the evolution of predictive models, and highlight the breakthrough AlphaFold provided the field.

Abstract SY45-02: Engineering strategies based on receptors derived from gdT cells

Abstract Gamma delta T (γδT) cells are a unique subset of T cells that play a crucial role in the body's immune response to infection, cancer, and cellular stress. Unlike their alpha-beta T cell counterparts, γδT cells can recognize and respond to a wide range of antigens without the need for antigen presentation by Major Histocompatibility Complex (MHC) molecules, allowing them to act swiftly in recognizing and eliminating abnormal cells. Clinical translation of γδT cell-based therapies, however, has faced significant challenges. Despite the safety profile of γδT cell infusions being well-established in cancer patients, the clinical outcomes have often been underwhelming (1). These results have raised questions about the efficacy of γδT cells in cancer treatment and the mechanisms through which they can be effectively harnessed. Despite these challenges, there have been encouraging developments that suggest the potential for γδT cell therapies. One notable advancement is the discovery of γδT cells carrying Chimeric Antigen Receptors (CARs) in long-term cancer survivors (2). These cells persist many years after treatment, indicating their long-lasting immune memory and surveillance capabilities. Another significant observation is the role of γδT cells as effective agents in immunotherapy that lack MHC class I molecules. This suggests that γδT cells can overcome the limitations of traditional immunotherapies that rely on MHC-mediated antigen presentation (3). Additionally, the successful use of engineered αβT cells expressing a high-affinity γδT Cell Receptor (TEG) in achieving complete remission in acute myeloid leukemia highlights the potential of redirecting T cell specificity to enhance anti-tumor responses (4). The exploration of Butyrophilin (BTN) and BTN-like (BTNL) molecules is a frontier in the understanding and enhancement of γδT cell therapies. BTN and BTNL molecules are implicated in the regulation of γδT cell responses through their interactions with γδT Cell Receptors (TCRs), particularly influencing the recognition of antigens and the subsequent immune response. Recent research focusing on BTN2 and BTN3 has aimed to delineate their roles in the activation and function of γδT cells, especially through γ9δ2TCRs (5). This research is crucial for unraveling the complexities of γδT cell antigen recognition and for developing strategies to enhance their therapeutic potential. Moreover, the diversity of the αβT cells, which are central to the TEG concept, presents another layer of complexity and opportunity in the design of T cell-based therapies. By engineering T cells to express specific, high-affinity γδTCRs, it is possible to harness the unique antigen-recognition capabilities of γδT cells while leveraging the proliferative and functional advantages of αβT cells. However detailed studies revealed a huge diversity in cell individual cell behavior (6). Finally, the role of costimulation in augmenting the activity of TEGs represents a promising area of investigation. Costimulatory signals are crucial for the full activation and sustained response of T cells. Enhancing costimulation in the context of TEGs could amplify their therapeutic efficacy, leading to improved outcomes in preclinical cancer treatment models by shaping the transcriptomic heterogeneity (7). In summary, while the clinical translation of γδT cell-based therapies has encountered challenges, recent discoveries and ongoing research offer hope for their future success. By understanding and manipulating the interactions between γδTCRs and BTN/BTNL molecules, diversifying TCR repertoires, and optimizing costimulatory signals, there is potential to unlock the full therapeutic promise of γδT cells in cancer immunotherapy. 1. Sebestyen Z, Prinz I, Dechanet-Merville J, Silva-Santos B, Kuball J. Translating gammadelta (gammadelta) T cells and their receptors into cancer cell therapies. Nat Rev Drug Discov. 2020;19(3):169-84. 2. Melenhorst JJ, Chen GM, Wang M, Porter DL, Chen C, Collins MA, et al. Decade-long leukaemia remissions with persistence of CD4(+) CAR T cells. Nature. 2022;602(7897):503-9. 3. Dart RJ, Zlatareva I, Vantourout P, Theodoridis E, Amar A, Kannambath S, et al. Conserved gammadelta T cell selection by BTNL proteins limits progression of human inflammatory bowel disease. Science. 2023;381(6663):eadh0301. 4. de Witte MAS, J.; 1, Weertman, N.; Daudeij, A.;, van der Wagen, L.; Oostvogels, R.; de Haar, C.; Prins, H.J.; Dohmen, W.W.C; Bartels-Wilmer,C.M.; Johanna, I; Sebestyen, Z.; Kuball, J.; Straetemans, T. First in human clinical responses and persistence data on TEG001: a next generation of engineered αβ T cells targeting AML and MM with a high affinity γ9δ2TCR. ASH confidential and under embargo until 04 11 2022 2022. 5. Mamedov MR, Vedova S, Freimer JW, Sahu AD, Ramesh A, Arce MM, et al. CRISPR screens decode cancer cell pathways that trigger gammadelta T cell detection. Nature. 2023;621(7977):188-95. 6. Dekkers JF, Alieva M, Cleven A, Keramati F, Wezenaar AKL, van Vliet EJ, et al. Uncovering the mode of action of engineered T cells in patient cancer organoids. Nat Biotechnol. 2023;41(1):60-9. 7. Hernandez-Lopez P, van Diest E, Brazda P, Heijhuurs S, Meringa A, Hoorens van Heyningen L, et al. Dual targeting of cancer metabolome and stress antigens affects transcriptomic heterogeneity and efficacy of engineered T cells. Nat Immunol. 2024;25(1):88-101. Citation Format: Jürgen Kuball. Engineering strategies based on receptors derived from gdT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY45-02.

Abstract 5006: Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines

Abstract The global surge in cancer cases, with a staggering 20 million new instances reported in 2023 alone, underscores an urgent demand for advanced and reliable cancer therapeutics. Immunotherapies centered around neoantigens have emerged as a promising avenue for enhancing treatment efficacy and have become a focal point in cancer research. We hypothesize that the tumor cells present a rich neoantigenic repertoire and the immunogenic neoantigens arising from the junctions of proteins translated from chimeric RNAs present viable targets for peptide and mRNA vaccines, offering a promising approach to impede tumor progression. To validate our hypothesis, we acquired a dataset of RNA fusions detected through RNAseq of tissue extracted from patient-derived xenograft (PDX) models established by XenoSTART, a global non clinical oncology contract research organization. This comprehensive dataset encompassed 985 PDX models across 27 cancer types, revealing three fusions associated with the neuregulin-1 (NRG1) gene. NRG1 gene fusions have been identified as a clinically actionable target for multiple solid tumors as they result in ErbB-mediated pathway activation. CD74-NRG1 has emerged as the most frequently reported NRG1 fusion across many cancers such as pancreatic ductal adenocarcinoma, triple negative breast cancer and ovarian cancer. The CD74-NRG1 fusion was identified in two PDX models harboring lung cancer (ST2891 and ST3204) and in an ovarian cancer model (ST088). The lung cancer PDXs displayed a notable abundance of fusion junction-crossing reads. We generated the CD74 (Exon 1-6) -NRG1 (Exon 6-12) fusion transcript model, where the first 6 exons of CD74 fused to exon 6 of NRG1 leading to an in-frame fusion. Validation of this fusion in PDX tissue was accomplished through PCR and Sanger sequencing of the PCR product. We assessed the affinity of the 9-mer neo peptide sequences formed by the translated junction of CD74-NRG1 to Major Histocompatibility Complex (MHC) Class I molecules using the in-silico prediction pipelines MHCNuggets and MixMHCPred-2 and found HLA-A*68:23 to be the MHC allele with highest binding affinity. HLA-Arena was used to investigate the binding affinity between the peptides and the MHC molecules via molecular docking, which revealed 4 peptides to be strong binders to HLA-A*68:23.The immunogenicity of these neoantigenic peptides to HLA-A*68:23 matched Peripheral Blood Mononuclear Cells (PBMCs) will be assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. To elucidate the specific T-cell clonotypes responding to the neo peptides, the 10X Genomics single-cell 5’ Gene Expression Assay, coupled with T Cell Receptor mapping, will be employed. The objective of the study is to establish a robust combinatorial therapeutic strategy leveraging peptide and mRNA vaccine technology to support an effective framework for personalized cancer treatment. Citation Format: Sakuni Rankothgedera, Micah Castillo, Shiyanth Thevasagayampillai, Cole Woody, Aaranyah Kandasamy, Armando Diaz, Michael Wick, Preethi Gunaratne. Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5006.

Exploiting Leishmania-Primed Dendritic Cells as Potential Immunomodulators of Canine Immune Response.

Dendritic cells (DCs) capture pathogens and process antigens, playing a crucial role in activating naïve T cells, bridging the gap between innate and acquired immunity. However, little is known about DC activation when facing Leishmania parasites. Thus, this study investigates in vitro activity of canine peripheral blood-derived DCs (moDCs) exposed to L. infantum and L. amazonensis parasites and their extracellular vesicles (EVs). L. infantum increased toll-like receptor 4 gene expression in synergy with nuclear factor κB activation and the generation of pro-inflammatory cytokines. This parasite also induced the expression of class II molecules of major histocompatibility complex (MHC) and upregulated co-stimulatory molecule CD86, which, together with the release of chemokine CXCL16, can attract and help in T lymphocyte activation. In contrast, L. amazonensis induced moDCs to generate a mix of pro- and anti-inflammatory cytokines, indicating that this parasite can establish a different immune relationship with DCs. EVs promoted moDCs to express class I MHC associated with the upregulation of co-stimulatory molecules and the release of CXCL16, suggesting that EVs can modulate moDCs to attract cytotoxic CD8+ T cells. Thus, these parasites and their EVs can shape DC activation. A detailed understanding of DC activation may open new avenues for the development of advanced leishmaniasis control strategies.

Helicobacter pylori enhances HLA-C expression in the human gastric adenocarcinoma cells AGS and can protect them from the cytotoxicity of natural killer cells.

Helicobacter pylori (H. pylori) seems to play causative roles in gastric cancers. H. pylori has also been detected in established gastric cancers. How the presence of H. pylori modulates immune response to the cancer is unclear. The cytotoxicity of natural killer (NK) cells, toward infected or malignant cells, is controlled by the repertoire of activating and inhibitory receptors expressed on their surface. Here, we studied H. pylori-induced changes in the expression of ligands, of activating and inhibitory receptors of NK cells, in the gastric adenocarcinoma AGS cells, and their impacts on NK cell responses. AGS cells lacked or had low surface expression of the class I major histocompatibility complex (MHC-I) molecules HLA-E and HLA-C-ligands of the major NK cell inhibitory receptors NKG2A and killer-cell Ig-like receptor (KIR), respectively. However, AGS cells had high surface expression of ligands of activating receptors DNAM-1 and CD2, and of the adhesion molecules LFA-1. Consistently, AGS cells were sensitive to killing by NK cells despite the expression of inhibitory KIR on NK cells. Furthermore, H. pylori enhanced HLA-C surface expression on AGS cells. H. pylori infection enhanced HLA-C protein synthesis, which could explain H. pylori-induced HLA-C surface expression. H. pylori infection enhanced HLA-C surface expression also in the hepatoma Huh7 and HepG2 cells. Furthermore, H. pylori-induced HLA-C surface expression on AGS cells promoted inhibition of NK cells by KIR, and thereby protected AGS cells from NK cell cytotoxicity. These results suggest that H. pylori enhances HLA-C expression in host cells and protects them from the cytotoxic attack of NK cells expressing HLA-C-specific inhibitory receptors.

Utilizing murine dendritic cell line DC2.4 to evaluate the immunogenicity of subunit vaccines in vitro.

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies.

Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis)

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Enhancing antigenic peptide discovery: Improved MHC-I binding prediction and methodology

The Major Histocompatibility Complex (MHC) is a critical element of the vertebrate cellular immune system, responsible for presenting peptides derived from intracellular proteins. MHC-I presentation is pivotal in the immune response and holds considerable potential in the realms of vaccine development and cancer immunotherapy. This study delves into the limitations of current methods and benchmarks for MHC-I presentation. We introduce a novel benchmark designed to assess generalization properties and the reliability of models on unseen MHC molecules and peptides, with a focus on the Human Leukocyte Antigen (HLA)–a specific subset of MHC genes present in humans. Finally, we introduce HLABERT, a pretrained language model that outperforms previous methods significantly on our benchmark and establishes a new state-of-the-art on existing benchmarks.

In silico evaluation of outer membrane protein S2 as a suitable vaccine candidate against Edwardsiella tarda infection of fish

AbstractObjectiveEdwardsiella tarda, the agent responsible for edwardsiellosis, is one of the primary emerging pathogens in fish aquaculture. The disease leads to significant economic loss for the farmers. Development of effective vaccines can minimize the disease burden.MethodsThe types of vaccinations that are currently at the center of the most significant research are subunit vaccines. Outer membrane proteins, which are a component of the bacteria, are very well known to be effective at stimulating immune responses in the host. In this study, the gene encoding for outer membrane protein S2 (OmpS2) of E. tarda was identified, cloned, and sequenced, followed by in silico analysis.ResultThe structure and subcellular localization of the protein were first confirmed. Homology modeling of the whole protein was performed, and the protein was checked for its eligibility as a vaccine candidate. This was followed by identifying antigenic sites, B‐cell epitopes, and cytotoxic T‐lymphocyte epitopes on OmpS2. We obtained a few distinct vaccine peptides from OmpS2. The complete genome of E. tarda was subjected to genome analysis to identify potential epitopes that would bind to the fish major histocompatibility complex molecule and elicit both humoral and cell‐mediated immune responses.ConclusionThis study provides valuable insights for consideration of OmpS2 as a potential vaccine candidate against E. tarda infection.

Evolutionary analysis of major histocompatibility complex variants in chytrid-resistant and susceptible amphibians

An amphibian emerging infectious disease (EID), chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), originated in Asia but primarily led to declines and extinctions in amphibian populations outside of Asia. Host major histocompatibility complex (MHC) molecules exhibit high polymorphism, and the evolution of MHC can be influenced by recombination and pathogens. Previous studies have indicated that host MHC class II is associated with Bd resistance. In this study, I conducted recombination and selection tests on functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) and an Australasian Bd-susceptible species (Litoria caerulea). Recombination at the same site was identified in both species, supporting the hypothesis that recombination contributes to MHC IIß1 diversity in amphibians. Positive selection was observed in MHC IIß1 alleles in both species. In L. caerulea, at least four amino acid sites were identified under significant positive selection in the MHC IIß1, whereas these sites were either negatively selected or conserved in B. gargarizans. This suggests these sites might be selected for Bd resistance. Hydrophobicity was detected in certain amino acid sites relating to Bd resistance, suggesting this physicochemical property may be a factor selected to counteract Bd infection. These findings of this study provide an evolutionary basis for understanding how amphibian MHC IIß1 may undergo selection in response to chytrid infection.

Virus-like particle-mediated delivery of structure-selected neoantigens demonstrates immunogenicity and antitumoral activity in mice

BackgroundNeoantigens are patient- and tumor-specific peptides that arise from somatic mutations. They stand as promising targets for personalized therapeutic cancer vaccines. The identification process for neoantigens has evolved with the use of next-generation sequencing technologies and bioinformatic tools in tumor genomics. However, in-silico strategies for selecting immunogenic neoantigens still have very low accuracy rates, since they mainly focus on predicting peptide binding to Major Histocompatibility Complex (MHC) molecules, which is key but not the sole determinant for immunogenicity. Moreover, the therapeutic potential of neoantigen-based vaccines may be enhanced using an optimal delivery platform that elicits robust de novo immune responses.MethodsWe developed a novel neoantigen selection pipeline based on existing software combined with a novel prediction method, the Neoantigen Optimization Algorithm (NOAH), which takes into account structural features of the peptide/MHC-I interaction, as well as the interaction between the peptide/MHC-I complex and the TCR, in its prediction strategy. Moreover, to maximize neoantigens’ therapeutic potential, neoantigen-based vaccines should be manufactured in an optimal delivery platform that elicits robust de novo immune responses and bypasses central and peripheral tolerance.ResultsWe generated a highly immunogenic vaccine platform based on engineered HIV-1 Gag-based Virus-Like Particles (VLPs) expressing a high copy number of each in silico selected neoantigen. We tested different neoantigen-loaded VLPs (neoVLPs) in a B16-F10 melanoma mouse model to evaluate their capability to generate new immunogenic specificities. NeoVLPs were used in in vivo immunogenicity and tumor challenge experiments.ConclusionsOur results indicate the relevance of incorporating other immunogenic determinants beyond the binding of neoantigens to MHC-I. Thus, neoVLPs loaded with neoantigens enhancing the interaction with the TCR can promote the generation of de novo antitumor-specific immune responses, resulting in a delay in tumor growth. Vaccination with the neoVLP platform is a robust alternative to current therapeutic vaccine approaches and a promising candidate for future personalized immunotherapy.

Asymmetric framework motion of TCRαβ controls load-dependent peptide discrimination.

Mechanical force is critical for the interaction between an αβ T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and β chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cβ FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαβ-pMHC agonist and antagonist complexes.

RPEMHC: improved prediction of MHC-peptide binding affinity by a deep learning approach based on residue-residue pair encoding.

Binding of peptides to major histocompatibility complex (MHC) molecules plays a crucial role in triggering T cell recognition mechanisms essential for immune response. Accurate prediction of MHC-peptide binding is vital for the development of cancer therapeutic vaccines. While recent deep learning-based methods have achieved significant performance in predicting MHC-peptide binding affinity, most of them separately encode MHC molecules and peptides as inputs, potentially overlooking critical interaction information between the two. In this work, we propose RPEMHC, a new deep learning approach based on residue-residue pair encoding to predict the binding affinity between peptides and MHC, which encode an MHC molecule and a peptide as a residue-residue pair map. We evaluate the performance of RPEMHC on various MHC-II-related datasets for MHC-peptide binding prediction, demonstrating that RPEMHC achieves better or comparable performance against other state-of-the-art baselines. Moreover, we further construct experiments on MHC-I-related datasets, and experimental results demonstrate that our method can work on both two MHC classes. These extensive validations have manifested that RPEMHC is an effective tool for studying MHC-peptide interactions and can potentially facilitate the vaccine development. The source code of the method along with trained models is freely available at https://github.com/lennylv/RPEMHC.

TCR Signals Controlling AdaptiveImmunity against Toxoplasmaand Cancer.

T cells play a crucial role in adaptive immunity by recognizing and eliminating foreign pathogens and abnormal cells such as cancer cells. T cell receptor (TCR), which is expressed on the surface of T cells, recognizes and binds to specific antigens presented by major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). This activation process leads to the proliferation and differentiation of T cells, allowing them to carry out their specific immune response functions. This chapter outlines the TCR signaling pathways that are common to different T cell subsets, as well as the recently elucidated TCR signaling pathway specific to CD8+ T cells and its role in controlling anti-Toxoplasma and anti-tumor immunity.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

Detection of Antigen Presentation by Murine Bone Marrow-Derived Dendritic Cells After Treatment with Nanoparticles.

Nanoparticles are frequently considered in vaccine applications due to their ability to co-deliver multiple antigens and adjuvants to antigen-presenting cells. Some nanoparticles also have intrinsic adjuvant properties that further enhance their ability to stimulate immune cells. The delivery of tumor-specific antigens to antigen-presenting cells (APCs) with subsequent antigenic peptide presentation in the context of class I major histocompatibility complex (MHC-I) molecules represents an essential effort in developing nanotechnology-based cancer vaccines. Experimental models are, therefore, needed to gauge the efficiency of nanotechnology carriers in achieving peptide antigen delivery to APCs and presentation in the context of MHC-I. The assay described herein utilizes a model antigen ovalbumin and model APCs, murine bone marrow-derived dendritic cells. The 25-D1.16 antibody, specific to the ovalbumin (OVA) MHC-I peptide SIINFEKL, recognizes this peptide presented in the context of the murine H2-Kb class I MHC molecule, allowing the presentation of this antigen on APCs to be detected by flow cytometry after nanoparticle delivery.