AMPD1 Is Associated With the Immune Response and Serves as a Prognostic Marker in HER2-Positive Breast Cancer

BackgroundAlthough immunotherapy has been used in the treatment of metastatic triple negative breast cancer (TNBC), its therapeutic influence on human epidermal growth factor receptor 2 (HER2)-positive subtype remains controversial. It is therefore imperative to find biomarkers that can predict the immune response in HER2+ BC.MethodsESTIMATE was utilized to compute the ImmuneScore and StromalScore from data obtained from TCGA database, and differentially expressed genes (DEGs) were identified. In addition, univariate Cox regression was used to assess candidate genes such as AMPD1, CD33, and CCR5. Gene set enrichment analysis (GSEA) was used to further understand AMPD1-associated pathways. Moreover, immunohistochemical analyses were performed to further reveal the relationship among AMPD1, CD4 and CD8 genes.ResultsThe expression of AMPD1 was markedly associated with disease outcome and tumor-infiltrating immune cells (TICs). In addition, AMPD1 was associated with lymph node status, age and the expression of PD-L1 and PD-L2. High AMPD1 expression was linked to longer overall survival (OS). Upregulated expression of AMPD1 correlated with the enrichment of immune-related signaling pathways. In addition, immunohistochemical analyses demonstrated a co-expression profile among AMPD1, CD4 and CD8 genes.ConclusionsTaken together, our data demonstrated that AMPD1 might serve as a novel biomarker for predicting the immune response and disease outcome in HER2+ BC.

Relationship between the rs333 Polymorphism in the CC Chemokine Receptor Type Five (CCR5) Gene and Immunological Disorders: Data from a Meta-Analysis

Introduction: Inflammatory Bowel Disease (IBD), periodontitis and Systemic Lupus Erythematous (SLE) are multifactorial diseases, one of the factors in the course of these diseases is the rs333 polymorphism in the CC chemokine receptor type five (CCR5) gene. However, the results remain contradictory. Therefore, we aimed to perform a meta-analysis evaluating the relation between this polymorphism and the aforementioned conditions. Material and Methods: A search in the literature was performed in diverse scientific and medical databases for studies published before June 22, 2020. The data were extracted from the studies and the statistical evaluation was performed by the calculations of statistical heterogeneity (I²), Odds Ratio (OR) with 95% of Confidence Intervals (CI) and publication bias. The values of P<0.05 were considered as significant for all calculations. Results: 19 articles with 21 case/control studies in 4,304 case patients and 3,492 controls were included. The meta-analysis showed a non-significant association among the rs333 polymorphism and IBD (OR = 1.05, 95% CI: 0.91-1.20, P = 0.51), periodontitis (OR = 0.86, 95% CI: 0.64-1.17, P = 0.34) or SLE (OR = 1.00, 95% CI: 0.56-1.80, P = 1.00) under the allelic model or for any other performed calculation. There were no obvious publication bias in the analyses. Conclusion: In conclusion, this current meta-analysis evidenced the non-significant relation among the rs333 polymorphism and the risk of IBD, periodontitis or SLE. Further studies are required to validate our data.

A Polymorphism in C-C Chemokine Receptor 5 (CCR5) Associates with Löfgren’s Syndrome and Alters Receptor Expression as well as Functional Response

C-C chemokine receptor 5 (CCR5) and polymorphisms in CCR5 gene are associated with sarcoidosis and Löfgren’s syndrome. Löfgren’s syndrome is an acute and usually self-remitting phenotype of sarcoidosis. We investigated whether the single nucleotide polymorphism (SNP) rs1799987 is associated with susceptibility for Löfgren’s syndrome and has an effect on CCR5 expression on monocytes and function of CCR5. A total of 106 patients with Löfgren’s syndrome and 257 controls were genotyped for rs1799987. Expression of CCR5 on monocytes was measured by flowcytometry. We evaluated calcium influx kinetics following stimulation upon N-formylmethionyl-leucyl-phenylalanine (fMLP) and macrophage inflammatory protein-1α (MIP-1α) on monocytes by measuring the median fluorescence intensity (MFI). The frequency of the G allele of rs1799987 was significantly higher in Löfgren’s syndrome than in healthy controls (p = 0.0015, confidence interval (CI) 1.22–2.32, odds ratio (OR) 1.680). Patients with a GG genotype showed higher CCR5 expression on monocytes than patients with the AA genotype (p = 0.026). A significantly (p = 0.027) lower count of patients with the GG genotype showed a calcium influx reaction to simulation upon MIP-1 α, compared with patients with the AA genotype. The rs1799987 G allele in CCR5 gene is associated with susceptibility to Löfgren’s syndrome and with quantitative and qualitative changes in CCR5, potentially effecting the inflammatory response.

First Survey of SNPs in TMEM154, TLR9, MYD88 and CCR5 Genes in Sheep Reared in Italy and their Association with Resistance to SRLVs Infection

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV), referred to as small ruminant lentiviruses (SRLVs), belong to the genus Lentivirus of the Retroviridae family. SRLVs infect both sheep and goats, causing significant economic losses and animal welfare damage. Recent findings suggest an association between serological status and allelic variants of different genes such as TMEM154, TLR9, MYD88 and CCR5. The aim of this work was to investigate the role of specific polymorphisms of these genes in SRLVs infection in some sheep flocks in Italy. In addition to those already known, novel variants in the TMEM154 (P7H, I74V, I105V) gene were detected in this study. The risk of infection was determined finding an association between the serological status and polymorphisms P7H, E35K, N70I, I74V, I105V of TMEM154, R447Q, A462S and G520R in TLR9 gene, H176H* and K190K* in MYD88 genes, while no statistical association was observed for the 4-bp deletion of the CCR5 gene. Since no vaccines or treatments have been developed, a genetically based approach could be an innovative strategy to prevent and to control SRLVs infection. Our findings are an important starting point in order to define the genetic resistance profile towards SRLVs infection.

Regulatory Noncoding and Predicted Pathogenic Coding Variants of CCR5 Predispose to Severe COVID-19

Genome-wide association studies (GWAS) found locus 3p21.31 associated with severe COVID-19. CCR5 resides at the same locus and, given its known biological role in other infection diseases, we investigated if common noncoding and rare coding variants, affecting CCR5, can predispose to severe COVID-19. We combined single nucleotide polymorphisms (SNPs) that met the suggestive significance level (P ≤ 1 × 10−5) at the 3p21.31 locus in public GWAS datasets (6406 COVID-19 hospitalized patients and 902,088 controls) with gene expression data from 208 lung tissues, Hi-C, and Chip-seq data. Through whole exome sequencing (WES), we explored rare coding variants in 147 severe COVID-19 patients. We identified three SNPs (rs9845542, rs12639314, and rs35951367) associated with severe COVID-19 whose risk alleles correlated with low CCR5 expression in lung tissues. The rs35951367 resided in a CTFC binding site that interacts with CCR5 gene in lung tissues and was confirmed to be associated with severe COVID-19 in two independent datasets. We also identified a rare coding variant (rs34418657) associated with the risk of developing severe COVID-19. Our results suggest a biological role of CCR5 in the progression of COVID-19 as common and rare genetic variants can increase the risk of developing severe COVID-19 by affecting the functions of CCR5.

Segmented intravaginal ring for the combination delivery of hydroxychloroquine and anti-CCR5 siRNA nanoparticles as a potential strategy for preventing HIV infection

Vaginal drug delivery has been shown to be a promising strategy for the prevention of sexually transmitted infections. Therapy delivered at the site of infection has many advantages including improved therapeutic efficacy, reduction in systemic toxicity, and reduced potential for development of drug resistance. We developed a “smart” combination intravaginal ring (IVR) that will (1) provide continuous release of hydroxychloroquine (HCQ) to induce T cell immune quiescence as the first-line of defense and (2) release nanoparticles containing anti-CCR5 siRNA only during sexual intercourse when triggered by the presence of seminal fluid as the second-line of defense. The IVR was capable of releasing HCQ over 25 days with a mean daily release of 31.17 ± 3.06 µg/mL. In the presence of vaginal fluid simulant plus seminal fluid simulant, over 12 × more nanoparticles (5.12 ± 0.9 mg) were released over a 4-h period in comparison to IVR segments that were incubated in the presence of vaginal fluid simulant alone (0.42 ± 0.19 mg). Anti-CCR5 siRNA nanoparticles were able to knockdown 83 ± 5.1% of CCR5 gene expression in vitro in the CD4+ T cell line Sup-T1. The IVR system also demonstrated to be non-cytotoxic to VK2/E6E7 vaginal epithelial cells. Graphical abstract

Infusion of CCR5 Gene-Edited T Cells Allows Immune Reconstitution, HIV Reservoir Decay, and Long-Term Virological Control

Antiretroviral therapy (ART) fails to fully restore immune function and is not curative. A single infusion of CCR5 gene-edited autologous CD4+ T cells (SB-728-T) led to sustained increases in CD4+ T cell counts, improved T cell homeostasis, and reduced the estimated size of the HIV reservoir. These outcomes were associated with the expansion and long-term persistence of a novel CCR5 gene-edited CD4+ T memory stem cell (CD45RAintROint TSCM) subset that can replenish the pool of more differentiated memory cells. We showed that novel CD45RAintROint TSCM cells are transcriptionally distinct from the previously described CD45RA+ TSCM and are minimally differentiated cells uncommitted to a specific Th-lineage. Subsequently, we showed in an independent trial that infusion of the SB-728-T cell product resulted in partial control of viral replication upon cessation of ART which was correlated with the frequencies of CCR5 gene-edited TSCM and their TEM progeny. Interestingly, one participant that remained off ART to this date demonstrated long-term maintenance of CCR5 gene-edited cells and increased frequency of polyfunctional HIV-specific CD4+ and CD8+ T cells, contributing to low levels of viral load 5 years post-infusion. Consequently, the generation of HIV protected memory CD4+ T cells by CCR5 disruption can contribute toward novel interventions aimed at achieving a sustained ART-free viral remission of HIV disease.

Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

Background Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. Methods A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. Results Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. Conclusions Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.