Akt1 and -2 inhibition diminishes terminal differentiation and enhances central memory CD8(+) T-cell proliferation and survival

The authors thank Dr Esteban Celis and Dr Rhea-Beth Markowitz for reviewing the manuscript and for their valuable suggestions and also thank Dr Lei Huang for his suggestions.Peer reviewedPublisher PD

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (<i>ACKR1</i>) / Duffy Antigen Receptor for Chemokines (<i>DARC</i>) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the <i>Duffy-Null</i> Allele

<div><p>The Atypical ChemoKine Receptor 1 (<i>ACKR1</i>) gene, better known as <i>Duffy</i> Antigen Receptor for Chemokines (<i>DARC</i> or <i>Duffy</i>), is responsible for the <i>Duffy</i> Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the <i>Duffy Null (Fy-)</i> allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of <i>DARC</i> which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific <i>Fy-</i> allele alters the expression pattern of <i>DARC</i> isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of <i>DARC</i> on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of <i>DARC</i> isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that <i>DARC</i> isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups.</p></div

The DARC gene structure and frequencies of the two major alleles; <i>Fy-</i> and <i>Fy</i>^<i>a/b</i> in HAPMAP experimental cohort compared to entire 1000 Genomes (1K) populations.

<p><b>(A).</b> Top, Model of DARC/ACKR1 gene structure indicating the two gene promoter-driven isoforms, DARC1/A and DARC2/B. Primers used for qPCR of transcript variants are indicated as double arrows. Also indicated are the <i>Duffy Null</i> allele (rs2814778) and <i>Fy</i>^<i>a/b</i> allele (rs12075). The two transcript products result in unique gene products; Duffy isoform A has 338aa and Duffy isoform B has 336aa. Top graph shows the allele frequencies of all documented polymorphisms in the DARC gene region, specifically in the ethnic cohorts of interest; African American (ASW), West African- Yoruba in Ibadan (YRI) and European American (CEPH). The overall ‘world’ variant frequencies are also indicated, summarizing all sampled populations in the 1000 Genomes. The bottom graph details the individual ancestry groups that encompass the global frequencies across the entire 1000 Genomes dataset for the entire DARC gene region. The X-axis matches the gene model with regard to relative gene structure location. The rs# IDs on the X axis of the bottom graph correspond to those on the top graph, for each SNP. The starred locus indicates a deletion polymorphism that resides in the promoter region of the DARC1/A isoform that has not previously been explicitly reported, described in the text (<i>rs17838198)</i>. This locus appears to have been under some selection influence in other populations, similar to the as the malaria selection influence on the <i>Duffy Null</i> allele in African populations, as it is the 3rd most prominent variant in the gene and prevalent in most populations. <b>(B).</b> The genotype frequencies for the <i>Fy-</i> and <i>Fy</i>^<i>a/b</i> loci in our cohort are shown in the inset panel on the left. The allele frequencies of <i>Fy-</i> and <i>Fy</i>^<i>a/b</i> for the entire corresponding ancestry 1K Genomes panels are shown in the inset panel on the right. The genotype percent distributions of the <i>Fy-</i> genotype across our West African—YRI, African American and European American subpopulations correlate with the high prevalence of the alleles in 1,000 Genome populations. Nearly half of the AA/ASW group is heterozygotes with an additional 40% being CC homozygotes, indicating more than 90% have the <i>Fy-</i> allele, as seen in the adjacent graph. The allele distribution table shows proportion values correlate with previously established numbers and validate our population.</p

DARC/ACKR1 protein is differentially expressed among lymphoblasts derived from divergent ancestry groups in HAPMAP/ 1,000 (1K) Genomes populations.

<p><b>(A).</b> Representative IHC images of DARC expression in the lymphoblasts of Africans (YRI) and European Americans (CEPH/CEU). DARC is stained with Vulcan Red chromogen nuclei are stained blue with hematoxylin. <b>(B).</b> Shows a distribution analysis of IHC scores across ancestry groups and <i>Fy-</i> genotypes. The full range of scores (0–4) were observed in our cohort, with clear trends within groups. European Americans were the only ancestry group to have a 0 score, indicating no DARC expression in these cells, correlating with lower transcription of the gene. Similarly, individuals with a homozygous <i>Duffy-positive</i> (TT) genotype were the only to have a score of 0, indicating that lowest levels of DARC expression in lymphoblast are associated with the European lineage and the TT genotype, in contrast to higher DARC expression in the lymphocytes of African lineages. Accordingly, the majority of high lymphoblast IHC scores (3 or 4) were in the African lineage. All African Americans only showed moderate expression levels in lymphoblasts.</p

Differential expression of DARC isoforms in lymphoblasts derived from 1K Genomes populations.

<p><b>(A)</b> Shows the relative expression levels of DARC 1 and DARC 2 transcripts among our HAPMAP cohort panel. The Fy- genotypes are indicated in the X axis of the top graph (A) and correlated HAPMAP cell line IDs are indicated in the X axis of the bottom graph (B). The “C” allele indicates the <i>Duffy Null</i> mutation. There is a clear trend of higher expression in the African and African Americans, with DARC 1 showing prominent expression. <b>(B)</b> Shows the relative ratios of DARC1/DARC2 isoforms in our cohort. The average ratio values for each ancestry category are shown as overlapping shaded box insets to display the values of each ancestry group. The highest ratio in the African category indicates the greatest difference between isoform expression values. C. ANOVA statistics indicate there are significant differences in DARC isoform transcripts across the cohort groups. The overall fitness across the entire group is mainly due to correlated expression of the isoforms for individuals with the C allele. There is clear trend of higher expression of the DARC 1 isoform in African and African American lineages, relative to European Americans. These data show a trend for lower expression in the European American (CEPH) categories and TT homozygotes for both DARC isoforms. These data indicate differences in isoform regulation, associated with the Fy- “<i>Duffy Null</i>” allele.</p

Statistical comparisons of relative levels of DARC isoform transcripts and ratios among ancestry and Fy- genotype groups.

<p>Statistical analyses across ancestry groups are on the left and genotype groups are on the right. ANOVA graphs indicate the individual group means (dotted blue line) of DARC isoform expression (top) and ratios (bottom) for stated categories. No statistically significant difference in individual transcript isoforms was identified across groups; however, significant differences were measured among ratios of DARC1/DARC2 isoforms. The means connection lines (solid blue and red) indicate the statistically significant comparisons. The significant p-values for pairwise T-test analyses of means is shown in the colors corresponding with each pair. The only statistically significant comparison across ancestry groups were between Amish and Yoruba groups. Across genotypes, there was a significant difference in DARC isoform ratios between TT genotype and both CT and CC genotypes. Significant differences between the isoform ratios indicate the isoforms are differentially regulated and the control of regulation is altered in isoform promoters with the C allele (Fy-).</p

DNA Damage Repair Classifier Defines Distinct Groups in Hepatocellular Carcinoma

DNA repair pathways have been associated with variability in hepatocellular carcinoma (HCC) clinical outcomes, but the mechanism through which DNA repair varies as a function of liver regeneration and other HCC characteristics is poorly understood. We curated a panel of 199 genes representing 15 DNA repair pathways to identify DNA repair expression classes and evaluate their associations with liver features and clinicopathologic variables in The Cancer Genome Atlas (TCGA) HCC study. We identified two groups in HCC, defined by low or high expression across all DNA repair pathways. The low-repair group had lower grade and retained the expression of classical liver markers, whereas the high-repair group had more clinically aggressive features, increased p53 mutant-like gene expression, and high liver regenerative gene expression. These pronounced features overshadowed the variation in the low-repair subset, but when considered separately, the low-repair samples included three subgroups: L1, L2, and L3. L3 had high DNA repair expression with worse progression-free (HR 1.24, 95% CI 0.81&ndash;1.91) and overall (HR 1.63, 95% CI 0.98&ndash;2.71) survival. High-repair outcomes were also significantly worse compared with the L1 and L2 groups. HCCs vary in DNA repair expression, and a subset of tumors with high regeneration profoundly disrupts liver biology and poor prognosis