Clinical Significance and Immunologic Landscape of a Five-IL(R)-Based Signature in Lung Adenocarcinoma

Interleukins (ILs) and interleukin receptors (ILRs) play important role in the antitumor immune response. However, the expression signature and clinical characteristics of the IL(R) family in lung adenocarcinoma (LUAD) remains unclear. The main purpose of this study was to explore the expression profile of IL(R) family genes and construct an IL(R)-based prognostic signature in LUAD. Five public datasets of 1,312 patients with LUAD were enrolled in this study. Samples from The Cancer Genome Atlas (TCGA) were used as the training set, and samples from the other four cohorts extracted from Gene Expression Omnibus (GEO) database were used as the validation set. Additionally, the profile of IL(R) family signature was explored, and the association between this signature and immunotherapy response was also analyzed. Meanwhile, the prognostic value was compared between this IL(R)-based signature and different immunotherapy markers. A signature based on five identified IL(R)s (IL7R, IL5RA, IL20RB, IL11, IL22RA1) was constructed using the TCGA dataset through univariate/multivariable Cox proportional hazards regression and least absolute shrinkage and selection operator (LASSO) Cox analysis. These cases with LUAD were stratified into high- and low-risk group according to the risk score. This signature showed a strong prognostic ability, which was verified by the five independent cohorts and clinical subtypes. The IL(R)-based models presented unique characteristics in terms of immune cell infiltration and immune inflammation profile in tumor microenvironment (TME). Biological pathway analysis confirmed that high-risk patients showed significant T- and B-cell immunosuppression and rapid tumor cell proliferation. More importantly, we researched the relationship between this IL(R)-based signature and immune checkpoints, tumor mutation burden (TMB), tumor purity and ploidy, and tumor immune dysfunction and exclusion (TIDE) score, which confirmed that this signature gave the best prognostic value. We first provided a robust prognostic IL(R)-based signature, which had the potential as a predictor for immunotherapy response to realize individualized treatment of LUAD.

Gene-Edited Interleukin CAR-T Cells Therapy in the Treatment of Malignancies: Present and Future

In recent years, chimeric antigen receptor T cells (CAR-T cells) have been faced with the problems of weak proliferation and poor persistence in the treatment of some malignancies. Researchers have been trying to perfect the function of CAR-T by genetically modifying its structure. In addition to the participation of T cell receptor (TCR) and costimulatory signals, immune cytokines also exert a decisive role in the activation and proliferation of T cells. Therefore, genetic engineering strategies were used to generate cytokines to enhance tumor killing function of CAR-T cells. When CAR-T cells are in contact with target tumor tissue, the proliferation ability and persistence of T cells can be improved by structurally or inductively releasing immunoregulatory molecules to the tumor region. There are a large number of CAR-T cells studies on gene-edited cytokines, and the most common cytokines involved are interleukins (IL-7, IL-12, IL-15, IL-18, IL-21, IL-23). Methods for the construction of gene-edited interleukin CAR-T cells include co-expression of single interleukin, two interleukin, interleukin combined with other cytokines, interleukin receptors, interleukin subunits, and fusion inverted cytokine receptors (ICR). Preclinical and clinical trials have yielded positive results, and many more are under way. By reading a large number of literatures, we summarized the functional characteristics of some members of the interleukin family related to tumor immunotherapy, and described the research status of gene-edited interleukin CAR-T cells in the treatment of malignant tumors. The objective is to explore the optimized strategy of gene edited interleukin-CAR-T cell function.

Interleukins and Interleukin Receptors Evolutionary History and Origin in Relation to CD4+ T Cell Evolution

Understanding the evolution of interleukins and interleukin receptors is essential to control the function of CD4+ T cells in various pathologies. Numerous aspects of CD4+ T cells’ presence are controlled by interleukins including differentiation, proliferation, and plasticity. CD4+ T cells have emerged during the divergence of jawed vertebrates. However, little is known about the evolution of interleukins and their origin. We traced the evolution of interleukins and their receptors from Placozoa to primates. We performed phylogenetic analysis, ancestral reconstruction, HH search, and positive selection analysis. Our results indicated that various interleukins' emergence predated CD4+ T cells divergence. IL14 was the most ancient interleukin with homologs in fungi. Invertebrates also expressed various interleukins such as IL41 and IL16. Several interleukin receptors also appeared before CD4+ T cells divergence. Interestingly IL17RA and IL17RD, which are known to play a fundamental role in Th17 CD4+ T cells first appeared in mollusks. Furthermore, our investigations showed that there is not any single gene family that could be the parent group of interleukins. We postulate that several groups have diverged from older existing cytokines such as IL4 from TGFβ, IL10 from IFN, and IL28 from BCAM. Interleukin receptors were less divergent than interleukins. We found that IL1R, IL7R might have diverged from a common invertebrate protein that contained TIR domains, conversely, IL2R, IL4R and IL6R might have emerged from a common invertebrate ancestor that possessed a fibronectin domain. IL8R seems to be a GPCR that belongs to the rhodopsin-like family and it has diverged from the Somatostatin group. Interestingly, several interleukins that are known to perform a critical function for CD4+ T cells such as IL6, IL17, and IL1B have gained new functions and evolved under positive selection. Overall evolution of interleukin receptors was not under significant positive selection. Interestingly, eight interleukin families appeared in lampreys, however, only two of them (IL17B, IL17E) evolved under positive selection. This observation indicates that although lampreys have a unique adaptive immune system that lacks CD4+ T cells, they could be utilizing interleukins in homologous mode to that of the vertebrates' immune system. Overall our study highlights the evolutionary heterogeneity within the interleukins and their receptor superfamilies and thus does not support the theory that interleukins evolved solely in jawed vertebrates to support T cell function. Conversely, some of the members are likely to play conserved functions in the innate immune system.

Experimental and population-genetic evidence for inflammation control functions of long noncoding RNAs and a novel tRNA-like transcript arising from the human NEAT1-MALAT1 genomic region

Background Uncontrolled inflammation is a key driver of atherosclerosis, myocardial infarction (MI), and multiple other diseases. Beyond proteins and microRNAs, long noncoding RNAs (lncRNAs) are implicated in inflammation control. We previously reported suppression of lncRNA NEAT1 in circulating immune cells of post-MI patients. In mice lacking lncRNAs NEAT1 or MALAT1 we observed major immune disturbances affecting monocyte-macrophage and T cell differentiation and rendering the immune system unstable and highly vulnerable to immune stress. Here, we report functions of a novel tRNA-type transcript arising from the NEAT1-MALAT1 gene cluster, and on genetic heterogeneity of this region in the human population. Methods and results While previously investigated mice were deficient in the entire NEAT1 or MALAT1 locus, we here aimed to selectively disrupted only the novel 59-nt tRNA-like transcript “menRNA” with hitherto unknown functions. Through CRISPR/Cas9 editing we developed 4 human THP-1 monocyte-macrophage cell line clones with deletions of different extension all of which prevented, however, normal transcript folding and formation of “menRNA”. Transcriptome mapping of all clones by RNA-sequencing identified dysregulation of innate immunity-related genes (IFI16, IFITM3, IRAK3, IRF2BP2, IRF3), chemokine and interleukin receptors (CCR10, IL11RA, IL12RB2, IL23A), cell surface receptors (CD37, CD40LG, CD72, FOCAD, ITGA6, MAEA, THY1), macrophage function-associated genes (ELANE, GRN, MIF, MMP25, MST1P2, PRTN3), tRNA-processing transcripts (GARS, QRSL1P3, QTRT1P1, THG1L, VARS), and small nucleolar RNAs (SNORA26.62.64, SNORD65.112). These data and functional assays indicate functions of NEAT1-derived “menRNA” distinct from those previously described for MALAT1-derived mascRNA. As multiple data suggest inflammation control functions of the NEAT1-MALAT1 region, we investigated the extent of genetic variability of this region in humans. In cohorts from the SHIP study coordinated by the Institute for Community Medicine Greifswald, screening of this region for sequence variants and possible phenotype associations was conducted the results of which are given in Figure 1. Consistent with prior findings, a MALAT1 SNP with very low minor allele frequency (MAF=0.01) was associated (p=0.0062) with systemic low level inflammation (CRP >3.0 mg/L). Unexpected was the association (p<0.01) of eight SNPs (low MAF=0.09 for all) with BMI >35 kg/m2 and LDL >164 mg/dl. Conclusions First, selective disruption of menRNA formation in human monocyte-macrophages provides evidence that this novel type of noncoding RNA has immunoregulatory functions. Second, the phenotype associations of SNPs within the NEAT1-MALAT1 gene cluster warrant further in-depth investigation of the molecular basis of these associations, and of their allele frequencies in cardiovascular disease patient cohorts. The first three and the last authors contributed equally to this work. Figure 1 Funding Acknowledgement Type of funding source: Other. Main funding source(s): “Transcriptome analysis of circulating immune cells to improve the assessment of prognosis and the response to novel anti-inflammatory treatments after myocardial infarction”; DZHK Shared Expertise project B19-006_SE FKZ 81X2100257

MYD88 L265P Elicits Mutation-specific Ubiquitination to Drive NF-κB Activation and Lymphomagenesis

Myeloid differentiation primary response protein 88 (MYD88) is a critical universal adapter that transduces signaling from the Toll-like receptors and interleukin receptors to downstream NF-kB. MYD88L265P (leucine changed to proline at position 265) is a gain-of-function mutation occurred frequently in B-cell malignancies such as Waldenstrom macroglobulinemia. Here we show that an E3 ligase RING finger protein family 138 (RNF138) catalyzed K63-linked non-proteolytic polyubiquitination of MYD88L265P, resulting in enhanced recruitment of interleukin 1 receptor-associated kinases and elevated NF-kB activation. However, RNF138 had little effect on wild-type MYD88 (MYD88WT). With either RNF138 knockdown or mutation on MYD88 ubiquitination sites, MYD88L265P was unable to constitutively activate NF-kB. A20, a negative regulator of NF-kB signaling, mediated K48-linked polyubiquitination of RNF138 for proteasomal degradation. Depletion of A20 further augmented MYD88L265P-mediated NF-kB activation and lymphoma growth. Furthermore, A20 expression was negatively correlated with RNF138 expression and NF-kB activation in lymphomas with MYD88L265P and in those without. Strikingly, RNF138 expression was positively correlated with NF-kB activation in lymphomas with MYD88L265P, but not in those without MYD88L265P. Collectively, our study reveals a novel mutation-specific biochemical reaction that drive B-cell oncogenesis, providing a therapeutic opportunity for targeting oncogenic MYD88L265P, while sparing MYD88WT that is critical to innate immunity.

QTY Code-designed Water-soluble Fc-fusion Cytokine Receptors Bind to their Respective Ligands

Cytokine release syndrome (CRS), or ‘cytokine storm’, is the leading side effect during chimeric antigen receptor (CAR)-T therapy that is potentially life-threatening. It also plays a critical role in viral infections such as Coronavirus Disease 2019 (COVID-19). Therefore, efficient removal of excessive cytokines is essential for treatment. We previously reported a novel protein modification tool called the QTY code, through which hydrophobic amino acids Leu, Ile, Val and Phe are replaced by Gln (Q), Thr (T) and Tyr (Y). Thus, the functional detergent-free equivalents of membrane proteins can be designed. Here, we report the application of the QTY code on six variants of cytokine receptors, including interleukin receptors IL4Rα and IL10Rα, chemokine receptors CCR9 and CXCR2, as well as interferon receptors IFNγR1 and IFNλR1. QTY-variant cytokine receptors exhibit physiological properties similar to those of native receptors without the presence of hydrophobic segments. The receptors were fused to the Fc region of immunoglobulin G (IgG) protein to form an antibody-like structure. These QTY code-designed Fc-fusion receptors were expressed in Escherichia coli and purified. The resulting water-soluble fusion receptors bind to their respective ligands with Kd values affinity similar to isolated native receptors. Our cytokine receptor–Fc-fusion proteins potentially serve as an antibody-like decoy to dampen the excessive cytokine levels associated with CRS and COVID-19 infection.

P001 PHARMACIST ROLE IN IBD MEDICATION OPTIMIZATION (PRIMO-IBD)

Introduction Inflammatory bowel disease (IBD) is a chronic disease state with growing focus, due to its significant medical and economic health burden. Although targeted therapy against tumor necrosis factor (anti-TNF), interleukin receptors, and integrin receptors have established roles in ulcerative colitis and Crohn’s disease management, medication accessibility and literacy contribute to medication non-adherence. Non-adherence to IBD therapy is associated with 62% higher costs for hospitalizations, along with increased disease mortality, relapse, loss of response, and antibody formation. IBD centers recognize the collaborative importance of a pharmacist’s role in medication optimization, patient adherence, and transitions of care. Methodology From November 2017 - April 2018, patients discharged from the inpatient IBD Service received a post discharge follow-up (PDFU) call from a pharmacist within 72 hours. Concurrently, the pharmacist provided pharmacotherapy optimization and education during the 4 hour weekly clinic visits, followed by a call within 72 hours. For all sites of care, the pharmacist performed a comprehensive evaluation for healthcare maintenance (including notable lab values, drug levels, and/or antibody levels), adherence to IBD medications, and drug interactions. The primary outcome was 30-day readmissions. The secondary outcomes included the number and severity of drug-related problems (DRPs) identified, validated by two gastroenterologists. Results 132 patients were included in the study (63 inpatient; 69 clinic). The inpatient 30-day readmission rate for the study period was 14.3% versus 22.1% in 2016 (P=0.15). In comparison, the 30-day hospitalization rate for clinic patients remained relatively unchanged. 123 DRPs were identified from 132 patients, averaging 0.93 DRPs per patient. There were 87 DRPs from the inpatient setting and 36 DRPs identified in clinic. Of the DRPs, 60% of DRPs were prescriber-related and 40% were patient-related. 2% (2 cases) were considered life-threatening, and 40% of cases were significant DRPs; the remaining DRPs were low risk. Potential admissions were avoided in 6 patients (11%) by early detection of a drug-related error. Conclusion Results demonstrate the opportunities for a pharmacist to be involved in managing biologics and health maintenance therapy in the IBD patient population. There was an overall positive trend of a pharmacist role on IBD admission rates and decrease in medication related errors.

A highly selective inhibitor of interleukin-1 receptor–associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase

Interleukin-1 receptor–associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor β–activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1–takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 μm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

A longitudinal study highlights shared aspects of the transcriptomic response to cardiogenic and septic shock

Background Septic shock (SS) and cardiogenic shock (CS) are two types of circulatory shock with a different etiology. Several studies have described the molecular alterations in SS patients, whereas the molecular factors involved in CS have been poorly investigated. We aimed to assess in the whole blood of CS and SS patients, using septic patients without shock (SC) as controls, transcriptomic modifications that occur over 1 week after ICU admission and are common to the two types of shock. Methods We performed whole blood RNA sequencing in 21 SS, 11 CS, and 5 SC. In shock patients, blood samples were collected within 16 h from ICU admission (T1), 48 h after ICU admission (T2), and at day 7 or before discharge (T3). In controls, blood samples were available at T1 and T2. Gene expression changes over time have been studied in CS, SS, and SC separately with a paired analysis. Genes with p value < 0.01 (Benjamini-Hochberg multiple test correction) were defined differentially expressed (DEGs). We used gene set enrichment analysis (GSEA) to identify the biological processes and transcriptional regulators significantly enriched in both types of shock. Results In both CS and SS patients, GO terms of inflammatory response and pattern recognition receptors (PRRs) were downregulated following ICU admission, whereas gene sets of DNA replication were upregulated. At the gene level, we observed that alarmins, interleukin receptors, PRRs, inflammasome, and DNA replication genes significantly changed their expression in CS and SS, but not in SC. Analysis of transcription factor targets showed in both CS and SS patients, an enrichment of CCAAT-enhancer-binding protein beta (CEBPB) targets in genes downregulated over time and an enrichment of E2F targets in genes with an increasing expression trend. Conclusions This pilot study supports, within the limits of a small sample size, the role of alarmins, PRRs, DNA replication, and immunoglobulins in the pathophysiology of circulatory shock, either in the presence of infection or not. We hypothesize that these genes could be potential targets of therapeutic interventions in CS and SS. Trial registration ClinicalTrials.gov, NCT02141607. Registered 19 May 2014.

Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.