Tumor antigen presentation and the associated signal transduction during carcinogenesis.

Numerous developments have been achieved in the study and treatment of cancer throughout the decades that it has been common. After decades of research, about 100 different kinds of cancer have been found, each with unique subgroups within certain organs. This has significantly expanded our understanding of the illness. A mix of genetic, environmental, and behavioral variables contribute to the complicated and diverse process of cancer formation. Mutations, or changes in the DNA sequence, are crucial to the development of cancer. These mutations have the ability to downregulate the expression and function of Major Histocompatibility Complex class I (MHC I) and MHCII receptors, as well as activate oncogenes and inactivate tumor suppressor genes. Cancer cells use this tactic to avoid being recognized by cytotoxic CD8+T lymphocytes, which causes issues with antigen presentation and processing. This review goes into great length into the PI3K pathway, changes to MHC I, and positive impacts of tsMHC-II on disease-free survival and overall survival and the involvement of dendritic cells (DCs) in different tumor microenvironments. The vital functions that the PI3K pathway and its link to the mTOR pathway are highlighted and difficulties in developing effective cancer targeted therapies and feedback systems has also been mentioned, where resistance mechanisms include RAS-mediated oncogenic changes and active PI3K signalling.

Proteasome isoforms in human thymi and mouse models.

The thymus is the organ where functional and self-tolerant T cells are selected through processes of positive and negative selection before migrating to the periphery. The antigenic peptides presented on MHC class I molecules of thymic epithelial cells (TECs) in the cortex and medulla of the thymus are key players in these processes. It has been theorized that these cells express different proteasome isoforms, which generate MHC class I immunopeptidomes with features that differentiate cortex and medulla, and hence positive and negative CD8+ T cell selection. This theory is largely based on mouse models and does not consider the large variety of noncanonical antigenic peptides that could be produced by proteasomes and presented on MHC class I molecules. Here, we review the multi-omics, biochemical and cellular studies carried out on mouse models and human thymi to investigate their content of proteasome isoforms, briefly summarize the implication that noncanonical antigenic peptide presentation in the thymus could have on CD8+ T cell repertoire and put these aspects in the larger framework of anatomical and immunological differences between these two species.

Humanization of Pan-HLA-DR mAb 44H10 Hinges on Critical Residues in the Antibody Framework.

Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions can also play essential roles in antigen binding. Here, we describe the humanization of the pan-HLA-DR mAb 44H10, a murine antibody displaying significant involvement of the framework region in antigen binding. Using a structure-guided approach, we identify and restore framework residues that directly interact with the antigen or indirectly modulate antigen binding by shaping the antibody paratope and engineer a humanized antibody with affinity, biophysical profile, and molecular binding basis comparable to that of the parental 44H10 mAb. As a humanized molecule, this antibody holds promise as a scaffold for the development of MHC class II-targeting therapeutics and vaccines.

Novel Methodology for the Design of Personalized Cancer Vaccine Targeting Neoantigens: Application to Pancreatic Ductal Adenocarcinoma.

Personalized cancer vaccines have emerged as a promising avenue for cancer treatment or prevention strategies. This approach targets the specific genetic alterations in individual patient's tumors, offering a more personalized and effective treatment option. Previous studies have shown that generalized peptide vaccines targeting a limited scope of gene mutations were ineffective, emphasizing the need for personalized approaches. While studies have explored personalized mRNA vaccines, personalized peptide vaccines have not yet been studied in this context. Pancreatic ductal adenocarcinoma (PDAC) remains challenging in oncology, necessitating innovative therapeutic strategies. In this study, we developed a personalized peptide vaccine design methodology, employing RNA sequencing (RNAseq) to identify prevalent gene mutations underlying PDAC development in a patient solid tumor tissue. We performed RNAseq analysis for trimming adapters, read alignment, and somatic variant calling. We also developed a Python program called SCGeneID, which validates the alignment of the RNAseq analysis. The Python program is freely available to download. Using chromosome number and locus data, SCGeneID identifies the target gene along the UCSC hg38 reference set. Based on the gene mutation data, we developed a personalized PDAC cancer vaccine that targeted 100 highly prevalent gene mutations in two patients. We predicted peptide-MHC binding affinity, immunogenicity, antigenicity, allergenicity, and toxicity for each epitope. Then, we selected the top 50 and 100 epitopes based on our previously published vaccine design methodology. Finally, we generated pMHC-TCR 3D molecular model complex structures, which are freely available to download. The designed personalized cancer vaccine contains epitopes commonly found in PDAC solid tumor tissue. Our personalized vaccine was composed of neoantigens, allowing for a more precise and targeted immune response against cancer cells. Additionally, we identified mutated genes, which were also found in the reference study, where we obtained the sequencing data, thus validating our vaccine design methodology. This is the first study designing a personalized peptide cancer vaccine targeting neoantigens using human patient data to identify gene mutations associated with the specific tumor of interest.

The DRB3 gene of the bovine major histocompatibility complex: discovery, diversity and distribution of alleles in commercial breeds of cattle and applications for development of vaccines.

The bovine Major Histocompatibility Complex (MHC), also known as the Bovine Leucocyte Antigen (BoLA) complex, is the genomic region that encodes the most important molecules for antigen presentation to initiate immune responses. The first evidence of MHC in bovines pointed to a locus containing 2 antigens, one detected by cytotoxic antiserum (MHC class I) and another studied by mixed lymphocyte culture tests (MHC class II). The most studied gene in the BoLA region is the highly polymorphic BoLA-DRB3, which encodes a ß chain with a peptide groove domain involved in antigen presentation for T cells that will develop and co-stimulate cellular and humoral effector responses. BoLA-DRB3 alleles have been associated with outcomes in infectious diseases such as mastitis, trypanosomiasis, and tick loads, and with production traits. To catalog these alleles, 2 nomenclature methods were proposed, and the current use of both systems makes it difficult to list, comprehend and apply these data effectively. In this review we have organized the knowledge available in all of the reports on the frequencies of BoLA-DRB3 alleles. It covers information from studies made in at least 26 countries on more than 30 breeds; studies are lacking in countries that are important producers of cattle livestock. We highlight practical applications of BoLA studies for identification of markers associated with resistance to infectious and parasitic diseases, increased production traits and T cell epitope mapping, in addition to genetic diversity and conservation studies of commercial and creole and locally adapted breeds. Finally, we provide support for the need of studies to discover new BoLA alleles and uncover unknown roles of this locus in production traits.

Preparation empty peptide-receptive MHC class I complex for large-scale detection through photolabile peptide ligands.

Peptide-major histocompatibility complex (pMHC) multimers are wide recognized as the premier technique for detecting, characterizing, and isolating antigen-specific CD8+ T-cell subsets. These multimers are specifically useful in studying infections, autoimmune conditions, and cancer through single-cell analysis techniques such as flow cytometry and fluorescence microscopy. However, the development of high-throughput assays with commercially available pMHC tetramers can be expensive, while in-house production may pose challenges for most biology research laboratories. In this context, we introduce a cost-friendly and uncomplicated protocol to prepare empty MHC class I tetramers using disulfide-stabilized molecules and photolabile peptide ligands. Our method relies on disulfide bond-stabilized MHC-I molecules, which demonstrated stability when folded into stable monomers in the presence of a photolabile epitope. These monomers, upon ultraviolet irradiation and streptavidin binding, efficiently assemble into tetramers devoid of any peptide. Following a short incubation with the peptide of interest under gentle conditions, the resulting pMHC tetramer effectively detects patient-sourced, neoantigen-specific T cells. Our unique approach streamlines large-scale pMHC generation, thus paving the way for advancements in T cell-based diagnostics and personalized therapies.

CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells.

The method presented herein is associated with the Lab Resource article titled "Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting" [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation.

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

Novel vaccination strategies based on optimal stimulation of CD4+ T helper cells for the treatment of oral squamous cell carcinoma.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor of the oral cavity. Despite recent advances in the field of oral cancer therapy, including the introduction of immunotherapeutic approaches, the 5-year survival rate remains steadily assessed around 50%. Thus, there is an urgent need for new therapeutic strategies. After the characterization of the immune phenotype of three human OSCC cell lines (CAL-27, SCC-25, and SCC-4) and one mouse OSCC cell line (MOC2) showing their similarities to resected patient tumors, we explored for the first time an experimental preclinical model of therapeutic vaccination with mouse OSCC MOC2 cell line stably expressing MHC class II antigens after CIITA gene transfection (MOC2-CIITA). Mice injected with MOC2-CIITA reject or strongly retard tumor growth; more importantly, vaccinated animals that fully reject MOC2-CIITA tumors display anti-tumor immunological memory protective against challenge with parental MOC2 tumor cells. Further experiments of adoptive cell transfer or in vivo cell depletion show that both CD4+ and CD8+ T lymphocytes prove fundamental in tumor rejection. This unprecedented approach for oral cancer opens the way for possible future translation of novel immunotherapeutic strategies to the human setting for the treatment of this tumor.

A Ménage à trois: NLRC5, immunity, and metabolism.

The nucleotide-binding and oligomerization domain-like receptors (NLRs) NLR family CARD domain-containing protein 5 (NLRC5) and Class II Major Histocompatibility Complex Transactivator (CIITA) are transcriptional regulators of major histocompatibility complex (MHC) class I and class II genes, respectively. MHC molecules are central players in our immune system, allowing the detection of hazardous 'non-self' antigens and, thus, the recognition and elimination of infected or transformed cells from the organism. Recently, CIITA and NLRC5 have emerged as regulators of selected genes of the butyrophilin (BTN) family that interestingly are located in the extended MHC locus. BTNs are transmembrane proteins exhibiting structural similarities to B7 family co-modulatory molecules. The family member BTN2A2, which indeed contributes to the control of T cell activation, was found to be transcriptionally regulated by CIITA. NLRC5 emerged instead as an important regulator of the BTN3A1, BTN3A2, and BTN3A3 genes. Together with BTN2A1, BTN3As regulate non-conventional Vγ9Vδ2 T cell responses triggered by selected metabolites of microbial origin or accumulating in hematologic cancer cells. Even if endogenous metabolites conform to the canonical definition of 'self', metabolically abnormal cells can represent a danger for the organism and should be recognized and controlled by immune system cells. Collectively, new data on the role of NLRC5 in the expression of BTN3As link the mechanisms regulating canonical 'non-self' presentation and those marking cells with abnormal metabolic configurations for immune recognition, an evolutionary parallel that we discuss in this perspective review.

Retinal vasculitis in HLA-A29 birdshot retinochoroiditis, particularities and imaging narrative of an under-estimated and diagnostic component of the disease.

BACKGROUND: HLA-A29 birdshot retinochoroiditis (BRC) is a primary stromal choroiditis (PSC), the hallmark being the choroidal rice-shaped hypopigmented fundus lesions ("birdshot lesions"). BRC is characterised by dual independent retinal vasculitis and choroiditis, the former often preceding manifest choroidal lesions. The purpose of this study was to analyse the type and severity of retinal vasculitis and determine whether it represented a diagnostic contribution. Medical records of patients with the diagnosis of BRC examined in the uveitis clinic of the Centre for Ophthalmic Specialised care (COS) in Lausanne from 1994 to 2020, were retrospectively reviewed. All patients had a complete ophthalmic examination, including visual field testing, optical coherence tomography (OCT), and fluorescein (FA) and indocyanine green (ICGA) angiography. Key retinal angiographic features were assessed. The study also established the angiographic score for retinal (FA) compared to choroidal involvement (ICGA). Among the 2102 newly diagnosed patients, 33 (1.57%) were diagnosed as BRC. Of the 21 patients with sufficient data included, all exhibited bilateral retinal vasculitis, of which 5 (24%) had no "birdshot lesions" at presentation with ICGA however always showing choroidal involvement. FA characteristics included (1) profuse retinal exudation in 17/21 cases (81%), (2) macular oedema in 17 patients (81%) with foveolar sparing for 14 of them (82%), (3) thick sheathing/staining of large posterior pole vessels in 13 patients (62%) and (4) profuse disc hyperfluorescence in all 21 patients. (5) A specific feature was the so-called pseudo arterio-venous circulatory delay in 17/21 cases (81%). The FA angiographic score at presentation was 14.49 ± 5.1 equivalent to the ICGA angiographic score of 14.29 ± 3.6, and higher than in other chorioretinitis entities. Both angiographic scores decreased similarly after treatment with a slower response of the retinal involvement. CONCLUSIONS: Retinal vasculitis in BRC is often very pronounced and presents distinct angiographic features that help substantially in the diagnosis and understanding of the disease course. Retinal vasculitis can present initially as an isolated feature in absence of the characteristic "birdshot lesions". The presence of all or some of the specific FA features strongly orient towards BRC to seek confirmation by ICGA and the search for the HLA-A29 antigen.

DNA structural features and variability of complete MHC locus sequences.

The major histocompatibility (MHC) locus, also known as the Human Leukocyte Antigen (HLA) genes, is located on the short arm of chromosome 6, and contains three regions (Class I, Class II and Class III). This 5 Mbp locus is one of the most variable regions of the human genome, yet it also encodes a set of highly conserved and important proteins related to immunological response. Genetic variations in this region are responsible for more diseases than in the entire rest of the human genome. However, information on local structural features of the DNA is largely ignored. With recent advances in long-read sequencing technology, it is now becoming possible to sequence the entire 5 Mbp MHC locus, producing complete diploid haplotypes of the whole region. Here, we describe structural maps based on the complete sequences from six different homozygous HLA cell lines. We find long-range structural variability in the different sequences for DNA stacking energy, position preference and curvature, variation in repeats, as well as more local changes in regions forming open chromatin structures, likely to influence gene expression levels. These structural maps can be useful in visualizing large scale structural variation across HLA types, in particular when this can be complemented with epigenetic signals.

Comprehensive alpha, beta, and delta cell transcriptomics reveal an association of cellular aging with MHC class I upregulation.

OBJECTIVES: This study aimed to evaluate the efficacy of a purification method developed for isolating alpha, beta, and delta cells from pancreatic islets of adult mice, extending its application to islets from newborn and aged mice. Furthermore, it sought to examine transcriptome dynamics in mouse pancreatic endocrine islet cells throughout postnatal development and to validate age-related alterations within these cell populations. METHODS: We leveraged the high surface expression of CD71 on beta cells and CD24 on delta cells to FACS-purify alpha, beta, and delta cells from newborn (1-week-old), adult (12-week-old), and old (18-month-old) mice. Bulk RNA sequencing was conducted on these purified cell populations, and subsequent bioinformatic analyses included differential gene expression, overrepresentation, and intersection analysis. RESULTS: Alpha, beta, and delta cells from newborn and aged mice were successfully FACS-purified using the same method employed for adult mice. Our analysis of the age-related transcriptional changes in alpha, beta, and delta cell populations revealed a decrease in cell cycling and an increase in neuron-like features processes during the transition from newborn to adult mice. Progressing from adult to old mice, we identified an inflammatory gene signature related to aging (inflammaging) encompassing an increase in ß-2 microglobulin and major histocompatibility complex (MHC) Class I expression. CONCLUSIONS: Our study demonstrates the effectiveness of our cell sorting technique in purifying endocrine subsets from mouse islets at different ages. We provide a valuable resource for better understanding endocrine pancreas aging and identified an inflammaging gene signature with increased ß-2 microglobulin and MHC Class I expression as a common hallmark of old alpha, beta, and delta cells, with potential implications for immune response regulation and age-related diabetes.

Identifying innovations produced by primary health care centers and evaluating their scalability: the SPRINT Occitanie cross-sectional study in France.

BACKGROUND: Practice-based research is one of the levers identified by the World Health Organization (WHO) to strengthen primary health care. The scaling of health and social care innovations has the potential to reduce inequities in health and to expand the benefits of effective innovations. It is now rapidly gaining the attention of decision-makers in health and social care, particularly in high-income countries. To meet the challenge of declining numbers of primary care physicians in France, Multi-professional Healthcare Centers (MHC) were created to bring together medical and paramedical professionals. They are a source of innovation in meeting the health challenges facing our populations. Specific methodology exists to identify health innovations and assess their scalability. A working group, including end-users and specialists, has adapted this methodology to the French context and the University department of general practice of Montpellier-Nîmes (France) launched a pilot study in Occitanie, a French region. OBJECTIVE: To identify and evaluate the scalability of innovations produced in pluri-professional healthcare centers in the Occitanie region. METHODS: A pilot, observational, cross-sectional study was carried out. The SPRINT Occitanie study was based on a questionnaire with two sections: MHC information and the modified Innovation Scalability Self-Administered Questionnaire (ISSaQ), version 2020. The study population was all 279 MHC in the Occitanie region. RESULTS: 19.3% (54) of MHC in the Occitanie region, responded fully or incompletely to the questionnaire. Four out of 5 U-MHCs were represented. Five MHC presented multiple innovations. The average per MHC was 1.94 (± 2.4) innovations. 26% of them (n = 9) had high scalability, 34% (n = 12) medium scalability and 40% (n = 14) low scalability. The main innovation represented (86%) were healthcare program, service, and tool. CONCLUSIONS: In our cross-sectional study, a quarter of the innovations were highly scalable. We were able to demonstrate the importance of MHC teams in working on primary care research through the prism of innovations. Primary-care innovations must be detected, evaluated, and extracted to improve their impact on their healthcare system.

Polygenic TB control and the sequence of innate/adaptive immune responses to infection: MHC-II alleles determine the size of the S100A8/9-producing neutrophil population.

Among several quantitative trait loci involved in tuberculosis (TB) control in mice, one was mapped within the chromosome 17 segment occupied by the H2 complex and another within the chromosome 3 segment comprising the S100A8/9 genes, which encode neutrophil inflammatory factor S100A8/9. Previously, we developed a panel of H2-congenic mouse strains differing by small segments of the major histocompatibility complex Class II (MHC-II) region from TB-susceptible H2j mice transferred onto the genetic background of the TB-resistant C57BL/6 (H2b) strain. Susceptible B6.I-9.3 mice differ from B6 progenitors by the alleles of their only classical MHC-II H2-Aß gene. The goals of the present study were to: (i) comprehensively characterise the differences in TB-related phenotypes between mice of the two strains and (ii) decipher interactions between the H2-Aß and S100A8/9 genes. Here, we describe the dynamics of TB-related phenotypes differentiating B6.I-9.3 and B6 mice (colony forming units counts, histopathology, lung immune cell infiltration and cytokine profiles). We show that disproportionally diminished CD4+ T-cell population, an enlarged S100A8/9-positive neutrophil population and higher S100A8/9 serum levels in B6.I-9.3 mice collectively form the 'susceptible' phenotype before infection. An increase in IL-17 and a decrease in intrferon-gamma production by CD4+ T-cells in these mice provide a mechanistic explanation of this phenotype. Using F2 segregation analysis, we show that the number of S100A8/9-producing neutrophils in lungs and spleens and the proportion of Th17 CD4+ T-cells in lungs are significantly lower in the presence of the MHC-II dominant 'resistant' b allele compared to the recessive 'susceptible' j/j genotype. This provides direct genetic evidence that MHC-II-regulated CD4+ T-cell landscapes determine neutrophil abundance before infection, an important pathogenic factor in TB immunity.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer.

Background: MHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. Methods: We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis. Results: Spatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1×10-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. Conclusions: Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent co-localization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.

Extracellular vesicle-packaged lncRNA from cancer-associated fibroblasts promotes immune evasion by downregulating HLA-A in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by immune evasion that contribute to poor prognosis. Cancer-associated fibroblasts (CAFs) play a pivotal role in orchestrating the PDAC tumour microenvironment. We investigated the role of CAF-derived extracellular vesicle (EV)-packaged long non-coding RNAs (lncRNAs) in immune evasion and explored gene therapy using engineered EVs loading small interfering RNAs (siRNAs) as a potential therapeutic strategy. Our findings highlight the significance of EV-packaged lncRNA RP11-161H23.5 from CAF in promoting PDAC immune evasion by downregulating HLA-A expression, a key component of antigen presentation. Mechanistically, RP11-161H23.5 forms a complex with CNOT4, a subunit of the mRNA deadenylase CCR4-NOT complex, enhancing the degradation of HLA-A mRNA by shortening its poly(A) tail. This immune evasion mechanism compromises the anti-tumour immune response. To combat this, we propose an innovative approach utilising engineered EVs as natural and biocompatible nanocarriers for siRNA-based gene therapy and this strategy holds promise for enhancing the effectiveness of immunotherapy in PDAC. Overall, our study sheds light on the critical role of CAF-derived EV-packaged lncRNA RP11-161H23.5/CNOT4/HLA-A axis in PDAC immune evasion and presents a novel avenue for therapeutic intervention.

Immunoinformatics and Reverse Vaccinology Approach for the Identification of Potential Vaccine Candidates against Vandammella animalimors.

Vandammella animalimorsus is a Gram-negative and non-motile bacterium typically transmitted to humans through direct contact with the saliva of infected animals, primarily through biting, scratches, or licks on fractured skin. The absence of a confirmed post-exposure treatment of V. animalimorsus bacterium highlights the imperative for developing an effective vaccine. We intended to determine potential vaccine candidates and paradigm a chimeric vaccine against V. animalimorsus by accessible public data analysis of the strain by utilizing reverse vaccinology. By subtractive genomics, five outer membranes were prioritized as potential vaccine candidates out of 2590 proteins. Based on the instability index and transmembrane helices, a multidrug transporter protein with locus ID A0A2A2AHJ4 was designated as a potential candidate for vaccine construct. Sixteen immunodominant epitopes were retrieved by utilizing the Immune Epitope Database. The epitope encodes the strong binding affinity, nonallergenic properties, non-toxicity, high antigenicity scores, and high solubility revealing the more appropriate vaccine construct. By utilizing appropriate linkers and adjuvants alongside a suitable adjuvant molecule, the epitopes were integrated into a chimeric vaccine to enhance immunogenicity, successfully eliciting both adaptive and innate immune responses. Moreover, the promising physicochemical features, the binding confirmation of the vaccine to the major innate immune receptor TLR-4, and molecular dynamics simulations of the designed vaccine have revealed the promising potential of the selected candidate. The integration of computational methods and omics data has demonstrated significant advantages in discovering novel vaccine targets and mitigating vaccine failure rates during clinical trials in recent years.