Targeted demethylation and activation of NLRC5 augment cancer immunogenicity through MHC class I.

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.

γδ T cells are effectors of immunotherapy in cancers with HLA class I defects.

DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3-5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of ß2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy.

Genetic Diversity and Sequence Conservation of Peptide-Binding Regions of MHC Class I Genes in Pig, Cattle, Chimpanzee, and Human.

Comparative analyses of MHC gene diversity and evolution across different species could offer valuable insights into the evolution of MHC genes. Intra- and inter-species sequence diversity and conservation of 12 classical major histocompatibility complex (MHC) class I genes from cattle, chimpanzees, pigs, and humans was analyzed using 20 representative allelic groups for each gene. The combined analysis of paralogous loci for each species revealed that intra-locus amino-acid sequence variations in the peptide-binding region (PBR) of MHC I genes did not differ significantly between species, ranging from 8.44% for SLA to 10.75% for BoLA class I genes. In contrast, intraspecies differences in the non-PBRs of these paralogous genes were more pronounced, varying from 4.59% for SLA to 16.89% for HLA. Interestingly, the Shannon diversity index and rate of nonsynonymous substitutions for PBR were significantly higher in SLA and BoLA than those in Patr and HLA. Analysis of peptide-binding pockets across all analyzed MHC class I genes of the four species indicated that pockets A and E showed the lowest and highest diversity, respectively. The estimated divergence times suggest that primate and artiodactyl MHC class I genes diverged 60.41 Mya, and BoLA and SLA genes diverged 35.34 Mya. These results offer new insights into the conservation and diversity of MHC class I genes in various mammalian species.

Cutting Edge: Unconventional CD8+ T Cell Recognition of a Naturally Occurring HLA-A*02:01-Restricted 20mer Epitope.

Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity.

Sequential modulation of the Wnt/ß-catenin signaling pathway enhances tumor-intrinsic MHC I expression and tumor clearance.

BACKGROUND: Progress in immunotherapy use for gynecologic malignancies is hampered by poor tumor antigenicity and weak T cell infiltration of the tumor microenvironment (TME). Wnt/ß-catenin pathway modulation demonstrated patient benefit in clinical trials as well as enhanced immune cell recruitment in preclinical studies. The purpose of this study was to characterize the pathways by which Wnt/ß-catenin modulation facilitates a more immunotherapy-favorable TME. METHODS: Human tumor samples and in vivo patient-derived xenograft and syngeneic murine models were administered Wnt/ß-catenin modulating agents DKN-01 and CGX-1321 individually or in sequence. Analytical methods included immunohistochemistry, flow cytometry, multiplex cytokine/chemokine array, and RNA sequencing. RESULTS: DKK1 blockade via DKN-01 increased HLA/MHC expression in human and murine tissues, correlating with heightened expression of known MHC I regulators: NFkB, IL-1, LPS, and IFNy. PORCN inhibition via CGX-1321 increased production of T cell chemoattractant CXCL10, providing a mechanism for observed increases in intra-tumoral T cells. Diverse leukocyte recruitment was noted with elevations in B cells and macrophages, with increased tumor expression of population-specific chemokines. Sequential DKK1 blockade and PORCN inhibition decreased tumor burden as evidenced by reduced omental weights. CONCLUSIONS: Wnt/ß-catenin pathway modulation increases MHC I expression and promotes tumor leukocytic infiltration, facilitating a pro-immune TME associated with decreased tumor burden. This intervention overcomes common tumor immune-evasion mechanisms and may render ovarian tumors susceptible to immunotherapy.

HLA Class I Analysis Provides Insight Into the Genetic and Epigenetic Background of Immune Evasion in Colorectal Cancer With High Microsatellite Instability.

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

Hormonal Regulation of the MHC Class I Gene in Thyroid Cells: Role of the Promoter "Tissue-Specific" Region.

In previous studies we have demonstrated that the expression of the Major Histocompatibility Complex (MHC) class I gene in thyrocytes is controlled by several hormones, growth factors, and drugs. These substances mainly act on two regions of the MHC class I promoter a "tissue-specific" region (-800 to -676 bp) and a "hormone/cytokines-sensitive" region (-500 to -68 bp). In a previous study, we have shown that the role of the "tissue-specific" region in the MHC class I gene expression is dominant compared to that of the "hormone/cytokines-sensitive" region. In the present report we further investigate the dominant role of the "tissue-specific" region evaluating the effect of thyroid stimulating hormone (TSH), methimazole (MMI), phenylmethimazole (C10), glucose and thymosin-α1. By performing experiments of electrophoretic mobility shift assays (EMSAs) we show that TSH, MMI and C10, which inhibit MHC class I expression, act on the "tissue-specific" region increasing the formation of a silencer complex. Glucose and thymosin-α1, which stimulate MHC class I expression, act decreasing the formation of this complex. We further show that the silencer complex is formed by two distinct members of the transcription factors families activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), c-jun and p65, respectively. These observations are important in order to understand the regulation of MHC class I gene expression in thyroid cells and its involvement in the development of thyroid autoimmunity.

Downregulation of Cell Surface Major Histocompatibility Complex Class I Expression Is Mediated by the Left-End Transcription Unit of Fowl Adenovirus 9.

Major histocompatibility complex class I (MHC-I) molecules play a critical role in the host's antiviral response by presenting virus-derived antigenic peptides to cytotoxic T lymphocytes (CTLs), enabling the clearance of virus-infected cells. Human adenoviruses evade CTL-mediated cell lysis, in part, by interfering directly with the MHC-I antigen presentation pathway through the expression of E3-19K, which binds both MHC-I and the transporter associated with antigen processing protein and sequestering MHC-I within the endoplasmic reticulum. Fowl adenoviruses have no homologues of E3-19K. Here, we show that representative virus isolates of the species Fowl aviadenovirus C, Fowl aviadenovirus D, and Fowl aviadenovirus E downregulate the cell surface expression of MHC-I in chicken hepatoma cells, resulting in 71%, 11%, and 14% of the baseline expression level, respectively, at 12 h post-infection. Furthermore, this work reports that FAdV-9 downregulates cell surface MHC-I through a minimum of two separate mechanisms-a lysosomal-independent mechanism that requires the presence of the fowl adenovirus early 1 (FE1) transcription unit located within the left terminal genomic region between nts 1 and 6131 and a lysosomal-dependent mechanism that does not require the presence of FE1. These results establish a new functional role for the FE1 transcription unit in immune evasion. These studies provide important new information about the immune evasion of FAdVs and will enhance our understanding of the pathogenesis of inclusion body hepatitis and advance the progress made in next-generation FAdV-based vectors.

BRAF mutations may identify a clinically distinct subset of glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Prior studies examining the mutational landscape of GBM revealed recurrent alterations in genes that regulate the same growth control pathways. To this regard, ~ 40% of GBM harbor EGFR alterations, whereas BRAF variants are rare. Existing data suggests that gain-of-function mutations in these genes are mutually exclusive. This study was designed to explore the clinical, pathological, and molecular differences between EGFR- and BRAF-mutated GBM. We reviewed retrospective clinical data from 89 GBM patients referred for molecular testing between November 2012 and December 2015. Differences in tumor mutational profile, location, histology, and survival outcomes were compared in patients with EGFR- versus BRAF-mutated tumors, and microarray data from The Cancer Genome Atlas was used to assess differential gene expression between the groups. Individuals with BRAF-mutant tumors were typically younger and survived longer relative to those with EGFR-mutant tumors, even in the absence of targeted treatments. BRAF-mutant tumors lacked distinct histomorphology but exhibited unique localization in the brain, typically arising adjacent to the lateral ventricles. Compared to EGFR- and IDH1-mutant tumors, BRAF-mutant tumors showed increased expression of genes related to a trophoblast-like phenotype, specifically HLA-G and pregnancy specific glycoproteins, that have been implicated in invasion and immune evasion. Taken together, these observations suggest a distinct clinical presentation, brain location, and gene expression profile for BRAF-mutant tumors. Pending further study, this may prove useful in the stratification and management of GBM.

Connecting MHC-I-binding motifs with HLA alleles via deep learning.

The selection of peptides presented by MHC molecules is crucial for antigen discovery. Previously, several predictors have shown impressive performance on binding affinity. However, the decisive MHC residues and their relation to the selection of binding peptides are still unrevealed. Here, we connected HLA alleles with binding motifs via our deep learning-based framework, MHCfovea. MHCfovea expanded the knowledge of MHC-I-binding motifs from 150 to 13,008 alleles. After clustering N-terminal and C-terminal sub-motifs on both observed and unobserved alleles, MHCfovea calculated the hyper-motifs and the corresponding allele signatures on the important positions to disclose the relation between binding motifs and MHC-I sequences. MHCfovea delivered 32 pairs of hyper-motifs and allele signatures (HLA-A: 13, HLA-B: 12, and HLA-C: 7). The paired hyper-motifs and allele signatures disclosed the critical polymorphic residues that determine the binding preference, which are believed to be valuable for antigen discovery and vaccine design when allele specificity is concerned.

Molecular Signatures of Natural Killer Cells in CMV-Associated Anterior Uveitis, A New Type of CMV-Induced Disease in Immunocompetent Individuals.

Cytomegalovirus (CMV) causes clinical issues primarily in immune-suppressed conditions. CMV-associated anterior uveitis (CMV-AU) is a notable new disease entity manifesting recurrent ocular inflammation in immunocompetent individuals. As patient demographics indicated contributions from genetic background and immunosenescence as possible underlying pathological mechanisms, we analyzed the immunogenetics of the cohort in conjunction with cell phenotypes to identify molecular signatures of CMV-AU. Among the immune cell types, natural killer (NK) cells are main responders against CMV. Therefore, we first characterized variants of polymorphic genes that encode differences in CMV-related human NK cell responses (Killer cell Immunoglobulin-like Receptors (KIR) and HLA class I) in 122 CMV-AU patients. The cases were then stratified according to their genetic features and NK cells were analyzed for human CMV-related markers (CD57, KLRG1, NKG2C) by flow cytometry. KIR3DL1 and HLA class I combinations encoding strong receptor-ligand interactions were present at substantially higher frequencies in CMV-AU. In these cases, NK cell profiling revealed expansion of the subset co-expressing CD57 and KLRG1, and together with KIR3DL1 and the CMV-recognizing NKG2C receptor. The findings imply that a mechanism of CMV-AU pathogenesis likely involves CMV-responding NK cells co-expressing CD57/KLRG1/NKG2C that develop on a genetic background of KIR3DL1/HLA-B allotypes encoding strong receptor-ligand interactions.

Natural Killer Cell Phenotype and Functionality Affected by Exposure to Extracellular Survivin and Lymphoma-Derived Exosomes.

The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-ß. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ.

Tumor Microenvironment Immune Response in Pancreatic Ductal Adenocarcinoma Patients Treated With Neoadjuvant Therapy.

BACKGROUND: Neoadjuvant folinic acid, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) and chemoradiation have been used to downstage borderline and locally advanced pancreatic ductal adenocarcinoma (PDAC). Whether neoadjuvant therapy-induced tumor immune response contributes to the improved survival is unknown. Therefore, we evaluated whether neoadjuvant therapy induces an immune response towards PDAC. METHODS: Clinicopathological variables were collected for surgically resected PDACs at the Massachusetts General Hospital (1998-2016). Neoadjuvant regimens included FOLFIRINOX with or without chemoradiation, proton chemoradiation (25 Gy), photon chemoradiation (50.4 Gy), or no neoadjuvant therapy. Human leukocyte antigen (HLA) class I and II expression and immune cell infiltration (CD4+, FoxP3+, CD8+, granzyme B+ cells, and M2 macrophages) were analyzed immunohistochemically and correlated with clinicopathologic variables. The antitumor immune response was compared among neoadjuvant therapy regimens. All statistical tests were 2-sided. RESULTS: Two hundred forty-eight PDAC patients were included. The median age was 64 years and 50.0% were female. HLA-A defects were less frequent in the FOLFIRINOX cohort (P = .006). HLA class II expression was lowest in photon and highest in proton patients (P = .02). The FOLFIRINOX cohort exhibited the densest CD8+ cell infiltration (P < .001). FOLFIRINOX and proton patients had the highest CD4+ and lowest T regulatory (FoxP3+) cell density, respectively. M2 macrophage density was statistically significantly higher in the treatment-naïve group (P < .001) in which dense M2 macrophage infiltration was an independent predictor of poor overall survival. CONCLUSIONS: Neoadjuvant FOLFIRINOX with or without chemoradiation may induce immunologically relevant changes in the tumor microenvironment. It may reduce HLA-A defects, increase CD8+ cell density, and decrease T regulatory cell and M2 macrophage density. Therefore, neoadjuvant FOLFIRINOX therapy may benefit from combinations with checkpoint inhibitors, which can enhance patients' antitumor immune response.

Thermostability profiling of MHC-bound peptides: a new dimension in immunopeptidomics and aid for immunotherapy design.

The features of peptide antigens that contribute to their immunogenicity are not well understood. Although the stability of peptide-MHC (pMHC) is known to be important, current assays assess this interaction only for peptides in isolation and not in the context of natural antigen processing and presentation. Here, we present a method that provides a comprehensive and unbiased measure of pMHC stability for thousands of individual ligands detected simultaneously by mass spectrometry (MS). The method allows rapid assessment of intra-allelic and inter-allelic differences in pMHC stability and reveals profiles of stability that are broader than previously appreciated. The additional dimensionality of the data facilitated the training of a model which improves the prediction of peptide immunogenicity, specifically of cancer neoepitopes. This assay can be applied to any cells bearing MHC or MHC-like molecules, offering insight into not only the endogenous immunopeptidome, but also that of neoepitopes and pathogen-derived sequences.

Genetics and the axial spondyloarthritis spectrum.

The axial SpAs (axSpAs) are clearly clinically a heterogeneous set of diseases with markedly varying extra-articular features. These diseases are all highly heritable and have overlapping but differing genetic origins. Shared features include association with HLA class I alleles and genes of the IL-23 pathway, among other things. Significant differences do exist however, both in the genetic loci involved and at specific loci in the individual genetic variants associated with each disease. These similarities and differences are of great interest in regards to disease pathogenesis and treatment development, although individually they are too small in effect to be of prognostic or diagnostic value. Polygenic risk scores, which capture a high proportion of the genetic variation between disorders, have been shown to have clinically useful discriminatory capacity in axSpA. This suggests they have the potential to enable improved disease classification, incorporating basic pathogenic features such as genomics, and ultimately benefitting clinical care. The aim of this article is to review the genetic characteristics of the spectrum of axSpAs and to discuss how this influences our understanding of the disease pathogenesis and the clinical implications of this understanding.

HLA Expression Correlates to the Risk of Immune Checkpoint Inhibitor-Induced Pneumonitis.

Tumor-infiltrating T cell rescue by programmed cell death receptor-1 (PD-1)/PD-1 ligand-1 (PD-L1) immune checkpoint blockade is a recommended treatment for malignant diseases, including metastatic non-small-cell lung cancer (mNSCLC), malignant melanoma (MM), head and neck, kidney, and urothelial cancer. Monoclonal antibodies (mAbs) against either PD-1 or PD-L1 are active agents for these patients; however, their use may be complicated by unpredictable immune-related adverse events (irAEs), including immune-related pneumonitis (IRP). We carried out a retrospective multi-institutional statistical analysis to investigate clinical and biological parameters correlated with IRP rate on a cohort of 256 patients who received real-world treatment with PD-1/PD-L1 blocking mAbs. An independent radiological review board detected IRP in 29 patients. We did not find statistical IRP rate correlation with gender, tumor type, specific PD-1 or PD-L1 blocking mAbs, radiation therapy, inflammatory profile, or different irAEs. A higher IRP risk was detected only in mNSCLC patients who received metronomic chemotherapy +/- bevacizumab compared with other treatments prior PD-1/PD-L1 blockade. Moreover, we detected a strong correlation among the IRP rate and germinal expression of HLA-B*35 and DRB1*11, alleles associated to autoimmune diseases. Our findings may have relevant implications in predicting the IRP rate in mNSCLC patients receiving PD-1/PD-L1 blockade and need to be validated on a larger patient series.

ASNEO: Identification of personalized alternative splicing based neoantigens with RNA-seq.

Cancer neoantigens have shown great potential in immunotherapy, while current software focuses on identifying neoantigens which are derived from SNVs, indels or gene fusions. Alternative splicing widely occurs in tumor samples and it has been proven to contribute to the generation of candidate neoantigens. Here we present ASNEO, which is an integrated computational pipeline for the identification of personalized Alternative Splicing based NEOantigens with RNA-seq. Our analyses showed that ASNEO could identify neopeptides which are presented by MHC I complex through mass spectrometry data validation. When ASNEO was applied to two immunotherapy-treated cohorts, we found that alternative splicing based neopeptides generally have a higher immune score than that of somatic neopeptides and alternative splicing based neopeptides could be a marker to predict patient survival pattern. Our identification of alternative splicing derived neopeptides would contribute to a more complete understanding of the tumor immune landscape. Prediction of patient-specific alternative splicing neopeptides has the potential to contribute to the development of personalized cancer vaccines.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.