Immune-modulating Genes Research Articles

Ameliorative role of camel protein hydrolysates diet against alkaline stress in Oreochrmis niloticus: Hematology, immune responses and their regulating genes expression, and histopathological assays.

This investigation looked at the ameliorative role of camel whey protein hydrolysates-diet (PH) in Oreochromis niloticus stocked under alkaline conditions. One hundred sixty fish (16.02 ± 0.14g) were allocated equally into four groups with four replications for 30 days. The first (control) and second (alkaline) groups were fed basal diets and maintained in fresh and alkaline water, respectively. The third and fourth groups were fed on a PH diet (basal diet containing 75g PH/kg) and maintained in fresh water and alkaline water, respectively. The hematology, immune-antioxidant indices, immune-regulatory genes, histopathological investigation of the spleen, and resistance to Aeromonas sobria were investigated. The results showed that the alkaline condition induced hematological disorders (lowered red blood cells, hemoglobin, packed cell volume, and white blood cell count) and immunosuppression (lowered phagocytic activity and index, lysozyme, nitric oxide, and complement 3) in the exposed fish. Alkaline exposure induced oxidative stress through elevation of the malondialdehyde and reduction in the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione S-reductase, and reduced glutathione). The immune modulatory genes (tolls like receptor-5, interleukin-1beta, interleukin-6, interleukin-8, interleukin-10, interleukin-17, nuclear factor kappa beta, and tumor necrosis factor-alpha) were down-regulated by exposure to alkaline conditions. The microscopic section of the spleen of the fish subjected to alkaline conditions showed notable hyperplasia of the melanomacrophage centers, besides vascular congestion, endothelial cell hypertrophy, and mild hypercellularity in the erythroid and lymphoid elements. In addition, few sections manifested more pronounced erythroid hyperplasia than the lymphoid one. The survival of the fish subjected to alkaline conditions was reduced during the A. sobria challenge. Feeding on a PH diet, the hematology was restored and the immune-antioxidant functions were modulated. Modulation of the immune-regulatory genes and increased survivability of the alkaline-exposed fish were noticed when fed on the PH diet. Consequently, we can recommend enriching the Nile tilapia diet with a 75g PH/kg diet especially when reared under alkaline conditions to support the immune functions of the fish.

IMMU-26. TARGETING CHK2-YBX1/3 INTERACTION TO OVERCOME IMMUNOSUPPRESSION IN GLIOMAS

Abstract Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cells are less studied. We performed an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cells. The screen revealed a DNA damage response gene-checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are in the clinical trials, in combination with PD-1/PD-L1 blockade, led to survival benefit in preclinical glioma models. Analysis of human GBMs demonstrated that Chek2 expression is inversely associated with antigen presentation and T-cell activation. However, the mechanism underlying the immunosuppressive function of Chek2 in gliomas is unknown. Here, we identified that glioma tumor cell intrinsic Chek2 interacts with Y-Box Binding Protein 1 and 3 (Ybx1&3) in ex vivo gliomas using immunoprecipitation followed by mass spectrometry. Independently, YBX3 was also identified as one of the most significantly phosphorylated protein upon Chek2 knockout using phosphoproteomics analysis. The YBX1&3 are DNA/RNA binding proteins implicated in transcriptional regulation of immune-modulatory genes. In silico docking using Alphafold and PIPER tool in Schrodinger revealed that the YBX1 cold-shock domain interacted near the ATP/ADP binding site in CHK2. This interface was also part of the CHK2-CHK2 dimer interface. Consistent with our model, disruption of CHK2 dimerization using prexasertib led to increased colocalization of CHK2-YBX1 in human GBM patient derived xenograft cells. Further, targeting of CHK2-YBX1 interaction with YBX1 inhibitor SU056 reversed the repression of IFNγ response genes in glioma cells. Together this study unveils a kinase independent (non-catalytic) function of CHK2, providing insight into the non-canonical immunosuppressive role of CHK2 in gliomas.

Comparison of Hypervirulent and Non-Hypervirulent Carbapenem-Resistant Acinetobacter baumannii Isolated from Bloodstream Infections: Mortality, Potential Virulence Factors, and Combination Therapy In Vitro.

Hypervirulent carbapenem-resistant Acinetobacter baumannii (hv-CRAB) has emerged in bloodstream infections (BSI). Cases of BSI caused by hv-CRAB (hv-CRAB-BSI) had posed a significant threat to hospitalized patients. In this study, 31 CRAB strains isolated from Chinese BSI patients were analyzed, of which 24 were identified as hv-CRAB-BSI and 7 as non-hv-CRAB-BSI, using the Galleria mellonella infection model. Patients with hv-CRAB-BSI had higher rates of septic shock (79.2% vs. 14.3%, p = 0.004) and mortality (66.7% vs. 14.3%, p = 0.028). All strains were resistant to most antibiotics but sensitive to colistin. Hv-CRAB-BSI showed lower resistance to minocycline than non-hv-CRAB-BSI (54.2% vs. 100%, p = 0.03). Whole-genome sequencing revealed that the detection rates of immune modulation genes ptk and epsA in hv-CRAB-BSI were significantly higher than in non-hv-CRAB-BSI (91.7% vs. 28.6%, p = 0.002). Additionally, all ST457 hv-CRAB-BSI lacked abaR, and all ST1486 non-hv-CRAB-BSI lacked adeG. The checkerboard dilution method assessed the efficacies of various antibiotic combinations, revealing that although synergism was rarely observed, the combination of colistin and minocycline showed the best efficacy for treating CRAB-BSI, regardless of whether the infections were hv-CRAB-BSI or non-hv-CRAB-BSI. These findings highlight the importance of analyzing molecular characteristics and exploring effective treatment strategies for hv-CRAB-BSI.

Genome-Wide Methylation Patterns in Primary Uveal Melanoma: Development of MethylSig-UM, an Epigenomic Prognostic Signature to Improve Patient Stratification.

Despite studies highlighting the prognostic utility of DNA methylation in primary uveal melanoma (pUM), it has not been translated into a clinically useful tool. We sought to define a methylation signature to identify newly diagnosed individuals at high risk for developing metastasis. Methylation profiling was performed on 41 patients with pUM with stage T2-T4 and at least three years of follow-up using the Illumina Infinium HumanMethylation450K BeadChip (N = 24) and the EPIC BeadChip (N = 17). Findings were validated in the TCGA cohort with known metastatic outcome (N = 69). Differentially methylated probes were identified in patients who developed metastasis. Unsupervised consensus clustering revealed three epigenomic subtypes associated with metastasis. To identify a prognostic signature, recursive feature elimination and random forest models were utilized within repeated cross-validation iterations. The 250 most commonly selected probes comprised the final signature, named MethylSig-UM. MethylSig-UM could distinguish individuals with pUM at diagnosis who develop future metastasis with an area under the curve of ~81% in the independent validation cohort, and remained significant in Cox proportional hazard models when combined with clinical features and established genomic biomarkers. Altered expression of immune-modulating genes were detected in MethylSig-UM positive tumors, providing clues for pUM resistance to immunotherapy. The MethylSig-UM model is available to enable additional validation in larger cohort sizes including T1 tumors.

TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo , TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.

Abstract SoA2-02: Implications of therapy induced senescence in breast cancer

Abstract The goal of breast cancer treatment is to cause cellular stress sufficient to kill and eliminate the tumor cells. Unfortunately, in many cases cancer cells survive and are not removed by surgery and thus can cause relapse and eventual mortality. Surviving cells have necessarily avoided both intrinsic (e.g., apoptosis) and extrinsic (e.g., immune surveillance) mechanisms of death. Studies over the last 20 years have shown that chemotherapy-treated breast cancer cells can evade cell death by activating programs of cellular senescence mediated by the tumor suppressor p53. While p53 can induce apoptosis in response to stress in some cell types, in breast cancer, p53 preferentially induces cell cycle arrest and senescence in response to chemotherapy. Data show that patients with breast cancers that are wild type for TP53 have a much lower probability of being eradicated by chemotherapy, and have a much shorter survival, compared to patients with TP53 mutant tumors. Human and mouse mammary tumors induce many markers of senescence after chemotherapy treatment, including those associated with cell cycle arrest and the senescence associated secretory phenotype (SASP). These arrested tumors evade death from mitotic catastrophe and can block apoptotic programs via elevated activity of BCL2 family members. More recent studies have addressed how induction of senescence deters immune surveillance. Breast cancer cells exposed to chemotherapy reorganize chromatin structure that amplifies stromal derived signals that stimulate expression of many immune genes, including checkpoint ligands such as PD-L1 and Galectin-9. Further, p53 can directly transactivate other immune modulatory genes such as CD80. Collectively, these properties of senescent cells enable their persistence following treatment and contribute to their potential to drive relapse. These same properties, however, can also be exploited as vulnerabilities to therapeutics such as senolytic drugs that target reliance on BCL2 family members or immunotherapies that target PD-L1 expression. Citation Format: J. Jackson. Implications of therapy induced senescence in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr SoA2-02.

Abstract 347: Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression

Abstract Background: TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting an unknown modulatory mechanism responsible for lack of TRAIL activity in vivo. Here, we describe that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu. Method: We performed RNAseq of MDA-MB-231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. RNAi, silencing key differentially regulated genes, along with RT-PCR, ELISA and CHIP assays, were used to validate RNAseq findings. Elucidation of the functional relevance of the outcome was supported both in vitro and in vivo by chemotaxis assay, cytotoxicity assay, RNAseq analysis of donor isolated neutrophils and intravital microscopy, CODEX analysis in TNBC xenografts from mice treated with the TRAIL respectively. Results: TRAIL treatment of the TNBC significantly induced expression of several cytokines, such as CXCLs 1, 2, 3, 8,11 and IL6, both in vitro and in vivo which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. GSEA of the RNAseq data indicated that NFKB pathway was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL in vitro and in vivo. However, the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs and IL6 significantly increased neutrophil chemotaxis. Additionally, CODEX analysis as well as intravital imaging confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Preincubation of neutrophils with supernatants from TRAIL-treated TNBC significantly inhibited their cytotoxic effect against TNBCs. Further, transcriptome analysis of neutrophils incubated with either TRAIL or supernatant of TRAIL treated TNBC revealed significant enrichment of expression of inflammatory cytokine genes, immune modulating and immune checkpoint genes like PDL1. Functional studies with these neutrophils confirmed their suppressive effect on T cell function as well as the effect of TRAIL on decreased neutrophil apoptosis. Conclusion: Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production promoting neutrophil chemotaxis and immune suppression. This study implies that alterations in the innate immune system may modulate the effects of TRAIL on TNBC tumors. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa Ann Ridnour, David A Wink, Yeap S. Ng, Roberto Weigert, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression [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 347.

Abstract A106: Single cell RNA sequencing of EPCAM+ cells reveals unique malignant biology

Abstract Pancreatic ductal adenocarcinoma (PDAC) is notable due to its complex and dense stroma which comprises a high proportion of non-malignant cells. Studying PDAC epithelial cells at the single cell level may yield insights which have not been appreciated through bulk tissue analysis. We analysed a novel single cell RNA sequencing (scRNA seq) dataset of 11000 epithelial (EPCAM+) cells from 7 resected PDAC samples and used a validation cohort of 43 samples from two published scRNA seq datasets. Malignant epithelial cells were identified by: gene expression, changes in copy number, and by the presence of known PDAC single nucleotide variants. This confirmed that sorting for EPCAM+ cells prior to scRNA seq resulted in an enriched proportion of malignant vs non-malignant tumor epithelial cells. This dataset of enriched single cell dataset was therefore useful for studying the influence of intrinsic malignant cell factors on PDAC biology. We observed that malignant cells do not segregate into classical or basal-like signatures, despite these being clinically validated bulk transcriptomic tumor subtypes. Indeed, classical and basal-like signatures module scores in PDAC in individual malignant cells were positively correlated, suggesting they are not divergent programs in malignant cells. Bulk transcriptomic signature subtypes are therefore most likely to reflect differences in PDAC tissue stroma, rather than in intrinsic malignant cell factors. Moreover, we sort to investigate intra- and inter- sample heterogeneity to understand the intrinsic malignant cell factors which may influence the biology of PDAC. We analysed single cell velocity to understand the dynamics of malignant cell states. This showed heterogeneity in expression of known pan-cancer malignant cell meta-programs which identified a population of cells with high expression of cell cycle genes, suggesting greater proliferation. We then explored expression of immune modulating genes to determine how malignant cells influence the immunosuppressive microenvironment. For example, we identified heterogeneity in HLA class 1 and antigen presentation gene expression which may be a mechanism of immune suppression in PDAC. Together these data demonstrate how studying scRNA seq of EPCAM+ cells reveals intrinsic malignant cell factors which cannot be appreciated through bulk RNA seq. Citation Format: Edward H. Arbe-Barnes, Ashwin Jainarayanan, Enas Abu-Shah, Rachael J.M. Bashford-Rogers, Shivan Sivakumar. Single cell RNA sequencing of EPCAM+ cells reveals unique malignant biology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A106.

The Role of Indoor Microbiome and Metabolites in Shaping Children's Nasal and Oral Microbiota: A Pilot Multi-Omic Analysis.

Maintaining a diverse and well-balanced nasal and oral microbiota is vital for human health. However, the impact of indoor microbiome and metabolites on nasal and oral microbiota remains largely unknown. Fifty-six children in Shanghai were surveyed to complete a questionnaire about their personal and environmental characteristics. The indoor microbiome and metabolites from vacuumed indoor dust were profiled via shotgun metagenomics and untargeted liquid chromatography-mass spectrometry (LC-MS). The nasal and oral microbiota in children was characterized using full-length 16S rRNA sequencing from PacBio. Associations between personal/environmental characteristics and the nasal/oral microbiota were calculated using PERMANOVA and regression analyses. We identified 6247, 431, and 342 microbial species in the indoor dust, nasal, and oral cavities, respectively. The overall nasal and oral microbial composition showed significant associations with environmental tobacco smoke (ETS) exposure during pregnancy and early childhood (p = 0.005 and 0.03, respectively), and the abundance of total indoor flavonoids and two mycotoxins (deoxynivalenol and nivalenol) (p = 0.01, 0.02, and 0.03, respectively). Notably, the abundance of several flavonoids, such as baicalein, eupatilin, isoliquiritigenin, tangeritin, and hesperidin, showed positive correlations with alpha diversity and the abundance of protective microbial taxa in nasal and oral cavities (p < 0.02), suggesting their potential beneficial roles in promoting nasal/oral health. Conversely, high carbohydrate/fat food intake and ETS exposure diminished protective microorganisms while augmenting risky microorganisms in the nasal/oral cavities. Further, potential microbial transfer was observed from the indoor environment to the childhood oral cavity (Moraxella catarrhalis, Streptococcus mitis, and Streptococcus salivarius), which could potentially increase virulence factors related to adherence and immune modulation and vancomycin resistance genes in children. This is the first study to reveal the association between the indoor microbiome/metabolites and nasal/oral microbiota using multi-omic approaches. These findings reveal potential protective and risk factors related to the indoor microbial environment.

Identification of immune‐related genes from the transcriptome of the venom gland in Cotesia plutellae

AbstractCotesia plutellae, an endoparasitoid wasp is the natural enemy against the diamondback moth, Plutella xylostella. Successful parasitization of C. plutellae requires maternal factors such as polydnavirus, venom proteins, and ovary proteins into the host, protecting their eggs from the host's immune system. To identify the immune‐related genes from the venom glands, we analyzed the transcriptome of venom glands via RNA‐seq. RNA‐seq analysis reveals that 39,207 transcripts were identified. Totally 106 immune‐related genes such as peptidoglycan‐recognition proteins, serpins, spätzles, phenoloxidase, defensin, and calreticulin, etc. were identified and classified into pathogen recognition, immune signal modulation, Toll pathway, IMD pathway, effectors, and other immune genes, depending on their biological functions. These results suggest that the transcriptome of the venom glands of C. plutellae can expand the understanding of insect immune response.

Canine amniotic membrane-derived mesenchymal stem cells ameliorate atopic dermatitis through regeneration and immunomodulation.

Mesenchymal stem cells (MSCs) are a promising tool for treating immune disorders. However, the immunomodulatory effects of canine MSCs compared with other commercialized biologics for treating immune disorders have not been well studied. In this study we investigated the characteristics and immunomodulatory effects of canine amnion membrane (cAM)-MSCs. We examined gene expression of immune modulation and T lymphocytes from activated canine peripheral blood mononuclear cell (PBMC) proliferation. As a result, we confirmed that cAM-MSCs upregulated immune modulation genes (TGF-β1, IDO1 and PTGES2) and suppressed the proliferation capacity of T cells. Moreover, we confirmed the therapeutic effect of cAM-MSCs compared with oclacitinib (OCL), the most commonly used Janus kinase (JAK) inhibitor, as a treatment for canine atopic dermatitis (AD) using a mouse AD model. As a result, we confirmed that cAM-MSCs with PBS treatment groups (passage 4, 6 and 8) compared with PBS only (PBS) though scores of dermatologic signs, tissue pathologic changes and inflammatory cytokines were significantly reduced. In particular, cAM-MSCs were more effective than OCL in the recovery of wound dysfunction, regulation of mast cell activity and expression level of immune modulation protein. Interestingly, subcutaneous injection of cAM-MSCs induced weight recovery, but oral administration of oclacitinib induced weight loss as a side effect. In conclusion, this study suggests that cAM-MSCs can be developed as a safe canine treatment for atopic dermatitis without side effects through effective regeneration and immunomodulation.

Recombinant strains of oncolytic vaccinia virus for cancer immunotherapy

Cancer virotherapy is an alternative therapeutic approach based on the viruses that selectively infect and kill tumor cells. Vaccinia virus (VV) is a member of the Poxviridae family of enveloped viruses with a large linear double-stranded DNA genome. The proven safety of VV strains as well as considerable transgene capacity of the viral genome, make VV an excellent platform for creating recombinant oncolytic viruses for cancer therapy. Furthermore, various genetic modifications can increase tumor selectivity and therapeutic efficacy of VV by arming it with the immune-modulatory genes or proapoptotic molecules, boosting the host immune system, and increasing cross-priming recognition of the tumor cells by T-cells or NK cells. In this review, we summarized the data on bioengineering approaches to develop recombinant VV strains for enhanced cancer immunotherapy.

Large-scale transcriptomic profiling of the tumor immune microenvironment in ALK+ lung cancer.

9020 Background: Patients (pts) with ALK+ Non-Small Cell Lung Cancer (NSCLC) do not derive significant clinical benefit from immune checkpoint inhibitors. To better understand this lack of immunotherapy sensitivity, we aimed to characterize major immune components of the tumor microenvironment (TME) by comprehensive transcriptomic and immunohistochemistry (IHC) analyses. Methods: We analyzed NGS data from 5490 NSCLC pts that underwent DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS) sequencing at Caris Life Sciences (Phoenix, AZ). 374 ALK-rearranged cases were evaluated, along with 3169 KRAS-mut ( STK11/ KEAP1-wt) and 1947 EGFR-mut cases serving as comparators with known heterogenous and inert immune TMEs, respectively. PD-L1 (22C3) was evaluated by IHC. Immune cell fractions were inferred using quanTIseq. Gene expression profiles were analyzed for a T cell-inflamed signature (TIS) predictive of response to immunotherapy and for other immune modulatory genes such as IFNG, GZMB, TGFB1, and those of the adenosine pathway ( CD73/NT5E, CD39/ENTPD1, ADORA1, ADORA2A/B). A significant difference between genomic subgroups was defined as fold-change &gt; 1.2. In an independent cohort of 14 ALK+ NSCLC pts, density and spatial organization of CD4+ and CD8+ T cells, Tregs, major myeloid lineage cells, PDL1, and CD73 were assessed by quantitative IHC. Results: ALK+ tumors were associated with high PD-L1 (≥50%) expression (40% vs 47% for KRAS-mut vs 18% for EGFR-mut, p &lt; 0.001) and low TMB (median 3 mut/MB vs 9 for KRAS-mut vs 4 for EGFR-mut, p &lt; 0.001). The abundance of CD8+ T cells (fold-change -1.3, p &lt; 0.001), Tregs (fold-change -1.2, p &lt; 0.001), M2 macrophages (fold-change 1.2, p &lt; 0.001), and CD4+ T cells (fold-change 1.9, p &lt; 0.001) differed from KRAS-mut; notably, similar to EGFR-mut. In ALK+ tumors, IFNG (fold-change -1.5, p &lt; 0.001), GZMB (fold-change -1.6, p &lt; 0.001), TGFB (fold-change -1.3, p &lt; 0.001), LAG-3 (fold-change -1.4 p &lt; 0.001), CD73/NT53 (fold-change -1.7 p &lt; 0.001) , and ADORA2A (fold-change -1.4, p &lt; 0.001) were decreased while ADORA1 (fold-change 1.3, p &lt; 0.001) was increased compared to KRAS-mut. EML4-ALK comprised 94.7% of the ALK+ tumors and distribution of EML4-ALK variants was consistent with prior reports (e.g. v1 35.6%, v3 35.1%). Immune cell fractions and immune-related gene expression did not vary significantly between major variant subgroups (v1 vs non-v1, and v3 vs non-v3, p &gt; 0.05). Conclusions: To our knowledge this is the largest transcriptomic analysis of the ALK+ NSCLC TME. Despite high levels of PD-L1, ALK+ tumors exhibit multiple features of an inert immune TME, primarily characterized by low TMB and decreased CD8+ T cells and immune activation markers. Our findings suggest that, while immunosuppressive factors such as M2 macrophages and adenosine signaling may also be targeted, strategies to enhance immunogenicity are critical for an effective immune response in ALK+ NSCLC.

Recombinant Strains of Oncolytic Vaccinia Virus for Cancer Immunotherapy.

Cancer virotherapy is an alternative therapeutic approach based on the viruses that selectively infect and kill tumor cells. Vaccinia virus (VV) is a member of the Poxviridae, a family of enveloped viruses with a large linear double-stranded DNA genome. The proven safety of the VV strains as well as considerable transgene capacity of the viral genome, make VV an excellent platform for creating recombinant oncolytic viruses for cancer therapy. Furthermore, various genetic modifications can increase tumor selectivity and therapeutic efficacy of VV by arming it with the immune-modulatory genes or proapoptotic molecules, boosting the host immune system, and increasing cross-priming recognition of the tumor cells by T-cells or NK cells. In this review, we summarized the data on bioengineering approaches to develop recombinant VV strains for enhanced cancer immunotherapy.

Dysregulation of PRMT5 in Chronic Lymphocytic Leukemia Supports Transformation to Diffuse Large B-Cell Lymphoma

Background: Richter's transformation (RT) is the progression of chronic lymphocytic leukemia (CLL) to a high-grade lymphoma, most commonly resembling a diffuse large B-cell-like lymphoma, that has a bleak prognosis of 6-12 months. Many of the recurrent somatic alterations identified in RT are also associated with the CLL-phase of disease, suggesting additional unidentified genetic, epigenetic, or molecular events underlie transformation. Aberrant PRMT5 expression is known to promote and maintain oncogenic signaling in non-Hodgkin's lymphomas but its role in CLL and RT remains unclear. Methods: Tissue microarrays and western blots were used to evaluate PRMT5 expression in CLL/SLL and RT lymph nodes (LN). Differentially expressed genes between patient-matched RT LNs and CLL PBMCs were identified with scRNA-seq & scV(D)J-seq. ScRNA-seq was utilized to identify transcriptional changes between Eμ-PRMT5/TCL1 & Eμ-TCL1 mice. Tumor allografts were generated by administering 5 x 10^6 CD19+CD5+ spleen cells from Eμ-PRMT5/TCL1 mice intravenously to C57/BL6J mice. One week post-engraftment mice were enrolled in treatment groups (PRT382 10mg/kg, n=10; vehicle, n=7) with all treatments administered for 4 contiguous days per week by oral gavage. Results: Tissue microarray analysis on 70 CLL and 15 RT LN cases showed robust PRMT5 staining in RT LNs and minimal staining in CLL tissues. Immunoblot analysis revealed that CLL cells from patients that eventually undergo transformation displayed significant increase in PRMT5 expression months prior to transformation, whereas CLL patients maintained variable to minimal PRMT5 expression. ScRNA-seq & scV(D)J-seq on RT LN biopsies and CLL PBMCs from two patients revealed that PRMT5 expression was restricted to distinct nodal B cell subpopulations co-expressing MYC, BIRC5, CD83, and CD69. One PRMT5+ cluster displayed overlapping expression of proliferative markers (Mki67, TOP2A, PCNA, CALM2/3, and HMGB1/2) with enriched EIF4 signaling. Anti-apoptotic and immune modulating genes (BCL21A, IL4I1, TCL1A, and CCL3/4) were enriched in the second PRMT5+ nodal cluster. Interestingly, transcriptional diversity was enhanced in clonally related RT B cell populations with an increased proportion of cells expressing PRMT5 when compared to the CLL-phase of disease. To further evaluate the role of PRMT5 in CLL disease progression, we conducted histopathologic analysis on the spleen and lymph nodes of 6 month old animals from Eµ-PRMT5/TCL1 & Eµ-TCL1 mouse models, revealing a RT-like histology with loss of germinal centers and effacement of normal architecture exclusively in Eµ-PRMT5/TCL1 animals. Transcriptional changes underlying phenotypic differences between Eµ-PRMT5/TCL1 & Eµ-TCL1 mice were also assessed by scRNA-seq, where splenic B cells of the Eµ-PRMT5/TCL1 exhibited increased expression of oncogenic and immune regulating genes (Myc, Mki67, Egr1, Cxcr5, Ccr7, Il-10, Ctla4, and Pd-L1). Interestingly, the dominant B-cell clusters enriched in Eµ-PRMT5/TCL1 spleen and LNs showed significant transcriptional overlap with human RT PRMT5 expressing LN clusters (e.g., Myc, Cd83, and Cd69). With the observation that PRMT5 may play a significant role in CLL-to-RT evolution, we then aimed to evaluate the use of targeted PRMT5 inhibition against murine RT-like tumors in vivo. Using the selective SAM-competitive PRMT5 inhibitor, PRT382, in Eµ-PRMT5/TCL1 allografts, we observed PRT382 treatment delayed leukemic expansion, increased median survival (p<0.0001), and reduced spleen mass compared to vehicle treated mice. Conclusion: We show that PRMT5 is upregulated in the lymph nodes of CLL patients prior to and after transformation, suggesting a role for PRMT5 in CLL transformation to RT. Likewise, the Eμ-PRMT5/TCL1 mouse model develops a RT-like histology and shows significant overlap in the transcriptional profile to human RT tumors. And further, inhibition of PRMT5 with PRT382 provides significant improvement in median overall survival and slows disease progression in Eμ-PRMT5/TCL1 adoptive transfer models. Our preclinical data identifies PRMT5 as a high-risk marker in CLL and provides rationale for the clinical evaluation of PRMT5 inhibition in the treatment of high-risk CLL/RT.

The effective factors in human-specific tropism and viral pathogenicity in orthopoxviruses.

The orthopoxvirus (OPV) genus includes several species that infect humans, including variola, monkeypox, vaccinia, and cowpox. Variola and monkeypox are often life-threatening diseases, while vaccinia and cowpox are usually associated with local lesions. The epidemic potential for OPVs may be lower than respiratory-borne viruses or RNA viruses. However, OPVs are notable for their spread and distribution in different environments and among different hosts. The emergence or re-emergence of OPVs in the human population can also occur in wild or domestic animals as intermediate hosts. More effective and safer vaccines for poxvirus can be developed by understanding how immunity is regulated in poxvirus and vaccines for DNA viruses. Downstream events in cells affected by the virus are regulated functionally by a series of characteristics that are affected by host cell interactions and responses of cells against viral infections, including the interferon pathway and apoptosis. Furthermore, infection outcome is greatly influenced by the distinct selection of host-range and immune-modulatory genes that confer the potential for pathogenesis and host-to-host transmission and the distinct host-range properties of each immune-modulatory gene. The present study reviewed the effective factors in human-restricted tropism and virus pathogenicity in OPVs.

Deep RNA sequencing of intensive care unit patients with COVID-19

COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host’s response in COVID-19. Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (> 100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies.

Spinal Cord Injury Impairs Lung Immunity in Mice.

Pulmonary infection is a leading cause of morbidity and mortality after spinal cord injury (SCI). Although SCI causes atrophy and dysfunction in primary and secondary lymphoid tissues with a corresponding decrease in the number and function of circulating leukocytes, it is unknown whether this SCI-dependent systemic immune suppression also affects the unique tissue-specific antimicrobial defense mechanisms that protect the lung. In this study, we tested the hypothesis that SCI directly impairs pulmonary immunity and subsequently increases the risk for developing pneumonia. Using mouse models of severe high-level SCI, we find that recruitment of circulating leukocytes and transcriptional control of immune signaling in the lung is impaired after SCI, creating an environment that is permissive for infection. Specifically, we saw a sustained loss of pulmonary leukocytes, a loss of alveolar macrophages at chronic time points postinjury, and a decrease in immune modulatory genes, especially cytokines, needed to eliminate pulmonary infections. Importantly, this injury-dependent impairment of pulmonary antimicrobial defense is only partially overcome by boosting the recruitment of immune cells to the lung with the drug AMD3100, a Food and Drug Administration-approved drug that mobilizes leukocytes and hematopoietic stem cells from bone marrow. Collectively, these data indicate that the immune-suppressive effects of SCI extend to the lung, a unique site of mucosal immunity. Furthermore, preventing lung infection after SCI will likely require novel strategies, beyond the use of orthodox antibiotics, to reverse or block tissue-specific cellular and molecular determinants of pulmonary immune surveillance.

Oligodendrocytes regulate the adhesion molecule ICAM-1 in neuroinflammation.

Recently, oligodendrocytes (Ol) have been attributed potential immunomodulatory effects. Yet, the exact mode of interaction with pathogenic CNS infiltrating lymphocytes remains unclear. Here, we attempt to dissect mechanisms of Ol modulation during neuroinflammation and characterize the interaction of Ol with pathogenic T cells. RNA expression analysis revealed an upregulation of immune-modulatory genes and adhesion molecules (AMs), ICAM-1 and VCAM-1, in Ol when isolated from mice undergoing experimental autoimmune encephalomyelitis (EAE). To explore whether AMs are involved in the interaction of Ol with infiltrating T cells, we performed co-culture studies on mature Ol and Th1 cells. Live cell imaging analysis showed direct interaction between both cell types. Eighty percentage of Th1 cells created contacts with Ol that lasted longer than 15 min, which may be regarded as physiologically relevant. Exposure of Ol to Th1 cells or their supernatant resulted in a significant extension of Ol processes, and upregulation of AMs as well as other immunomodulatory genes. Our observations indicate that blocking of oligodendroglial ICAM-1 can reduce the number of Th1 cells initially contacting the Ol. These results suggest that AMs may play a role in the interaction between Ol and Th1 cells. We identified Ol interacting with CD4+ cells in vivo in spinal cord tissue of EAE diseased mice indicating that our in vitro findings are of interest to further scientific research in this field. Further characterization and understanding of Ol interaction with infiltrating cells may lead to new therapeutic strategies enhancing Ol protection and remyelination potential. Oligodendrocytes regulate immune modulatory genes and adhesion molecules during autoimmune neuroinflammation Oligodendrocytes interact with Th1 cells in vitro in a physiologically relevant manner Adhesion molecules may be involved in Ol-Th1 cell interaction.

Identification of loci associated with susceptibility to Mycobacterium avium subsp. paratuberculosis infection in Holstein cattle using combinations of diagnostic tests and imputed whole-genome sequence data.

Bovine paratuberculosis (PTB) is a chronic inflammatory disease caused by Mycobacterium avium susbp. paratuberculosis (MAP). Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) significantly associated with susceptibility to bovine PTB. The main objective of this study was to identify quantitative trait loci (QTLs) associated with MAP infection in Spanish Holstein cows (N = 983) using combinations of diagnostic tests and imputed whole-genome sequence (WGS) data. The infection status of these animals was defined by three diagnostic methods including ELISA for MAP-antibodies detection, and tissue culture and PCR for MAP detection. The 983 cows included in this study were genotyped with the Bovine MD SNP50 Bead Chip, and the corresponding genotypes were imputed to WGS using the 1,000 Bull genomes reference population. In total, 33.77 million SNP variants per animal were identified across the genome. Linear mixed models were used to calculate the heritability (h2) estimates for each diagnostic test and test combinations. Next, we performed a case-control GWAS using the imputed WGS datasets and the phenotypes and combinations of phenotypes with h2 estimates > 0.080. After performing the GWAS, the test combinations that showed SNPs with a significant association (PFDR ≤ 0.05), were the ELISA-tissue PCR-tissue culture, ELISA-tissue culture, and ELISA-tissue PCR. A total of twelve quantitative trait loci (QTLs) highly associated with MAP infection status were identified on the Bos taurus autosomes (BTA) 4, BTA5, BTA11, BTA12, BTA14, BTA23, BTA24, and BTA28, and some of these QTLs were linked to immune-modulating genes. The identified QTLs on BTA23 spanning from 18.81 to 22.95 Mb of the Bos taurus genome overlapped with several QTLs previously found to be associated with PTB susceptibility, bovine tuberculosis susceptibility, and clinical mastitis. The results from this study provide more clues regarding the molecular mechanisms underlying susceptibility to PTB infection in cattle and might be used to develop national genetic evaluations for PTB in Spain.