DNA Methylation Maintains Allele-specific KIR Gene Expression in Human Natural Killer Cells

Killer immunoglobulin-like receptors (KIR) bind self–major histocompatibility complex class I molecules, allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes, but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression, 3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5′ gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor, 5-aza-2′-deoxycytidine, induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus, NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles

Inhibitors of the CD73-adenosinergic checkpoint as promising combinatory agents for conventional and advanced cancer immunotherapy

The cell surface enzyme CD73 is increasingly appreciated as a pivotal non-redundant immune checkpoint (IC) in addition to PD-1/PD-L1 and CTLA-4. CD73 produces extracellular adenosine (eADO), which not only inhibits antitumor T cell activity via the adenosine receptor (AR) A2AR, but also enhances the immune inhibitory function of cancer-associated fibroblasts and myeloid cells via A2BR. Preclinical studies show that inhibition of the CD73-adenosinergic pathway in experimental models of many solid tumors either as a monotherapy or, more effectively, in combination with PD-1/PD-L1 or CTLA-4 IC blockades, improves antitumor immunity and tumor control. Consequently, approximately 50 ongoing phase I/II clinical trials targeting the CD73-adenosinergic IC are currently listed on https://clinicaltrials.gov. Most of the listed trials employ CD73 inhibitors or anti-CD73 antibodies alone, in combination with A2AR antagonists, and/or with PD-1/PD-L1 blockade. Recent evidence suggests that the distribution of CD73, A2AR and A2BR in tumor microenvironments (TME) is heterogeneous, and this distribution affects CD73-adenosinergic IC function. The new insights have implications for the optimally effective, carefully tailored approaches to therapeutic targeting of this essential IC. In the mini-review, we briefly discuss the cellular and molecular mechanisms of CD73/eADO-mediated immunosuppression during tumor progression and therapy in the spatial context of the TME. We include preclinical data regarding therapeutic CD73-eADO blockade in tumor models as well as available clinical data from completed trials that targeted CD73-adenosinergic IC with or without PD-1/PD-L1 inhibitors and discuss factors that are potentially important for optimal therapeutic outcomes in cancer patients

Periluminal Distribution of HIV-Binding Target Cells and Gp340 in the Oral, Cervical and Sigmoid/Rectal Mucosae: A Mapping Study

<div><p>Studies have shown that the transmission of HIV is most likely to occur via rectal or vaginal routes, and rarely through oral exposure. However, the mechanisms of virus entry at mucosal surfaces remain incompletely understood. Prophylactic strategies against HIV infection may be attainable once gaps in current knowledge are filled. To address these gaps, we evaluated essentially normal epithelial surfaces and mapped the periluminal distribution of CD4⁺ HIV target cells, including T cells and antigen-presenting cells, and an HIV-binding molecule gp340 that can be expressed by epithelial cells in secreted and cell-associated forms. Immunohistochemistry for CD4, CD16, CD3, CD1a and gp340 in human oral, rectal/sigmoid and cervical mucosal samples from HIV-negative subjects demonstrated that periluminal HIV target cells were more prevalent at rectal/sigmoid and endocervical surfaces lined by simple columnar epithelium, than at oral and ectocervical surfaces covered by multilayered stratified squamous epithelium (p<0.001). gp340 expression patterns at these sites were also distinct and strong in oral minor salivary gland acini and ducts, including ductal saliva, in individual rectum/sigmoid and endocervix periluminar columnar cells, and in ectocervix squamous cells. Only weak expression was noted in the oral non-ductal squamous epithelium. We conclude that periluminal HIV target cells, together with periluminal epithelial cell-associated gp340 appear to be most accessible for HIV transmission at rectal/sigmoid and endocervical surfaces. Our data help define vulnerable structural features of mucosal sites exposed to HIV.</p></div

Schematic diagrams summarizing the observed results.

<p>The model maps gp340 and HIV target cells at the normal mucosal surface portals. The model proposes that HIV target cells and the epithelial cell-associated alternative HIV-binding molecule gp340 rarely co-localize at the luminal surfaces lined by squamous epithelium, either oral or ectocervical, largely because the target cells typically stay away from the periluminal layer <b>(A,B)</b>. Moreover, expression of gp340 in oral squamous epithelium appears to depend largely upon salivary glands, suggesting that it is primarily soluble <b>(A)</b>, while the ectocervial squamous epithelium often showed strong gp340 expression throughout <b>(B)</b>, without evidence of gp340-secreting glands. In contrast, HIV target cells and cell-associated gp340 frequently, but not always, co-localized periluminally in simple columnar epithelia of the colon/rectum and endocervix <b>(C, D)</b>.</p

Distribution of CD4⁺ and CD16⁺ cells in squamous epithelia of the oral and ectocervical mucosae and in simple columnar epithelia of the rectum/sigmoid and endocervix.

<p>Tissue sections were stained by standard IHC for CD4 (Panels <b>A,C,E,G</b>) or CD16 (Panels <b>B,D,F,H</b>), as described in Materials and Methods. Representative images are shown. Receptor-positive cells appear brown and cell nuclei appear blue. Arrows indicate a few examples of positive cells located within the epithelium. Small round CD4^bright cells (black arrows) are T cells, while antigen-presenting cells (APC) are larger and CD4^dim (yellow arrows). Note that CD4⁺ cells are typically present several cell layers deep within the stratified squamous epithelium and lamina propria of oral (<b>A</b>) and ectocervical mucosae (<b>C</b>), similar to CD16⁺ APC (<b>B</b> and <b>D</b>, respectively). Small round CD4^bright lymphocytes, CD4^dim APC and CD16⁺ APC are also present within the simple columnar epithelia (<b>E-H</b>). Note the horizontal orientation of CD16⁺ APC at the base of columnar cells in the endocervix (<b>F</b>). Also note that some CD16⁺ APC in the rectal epithelium are oriented vertically with dendritic processes reaching the luminal surface (insert, <b>H</b>). Bar = 100 μm.</p

Comparison of periluminal CD4⁺ (A) and CD16⁺ (B) counts at all sites.

<p>Stained and photographed sections (200x) were used to record the CD4⁺ and CD16⁺ cells, respectively, in the periluminal layer of squamous epithelia and in single-layer simple columnar epithelia. Charts show respective cell counts per field by region, each data point is one field. Mann Whitney tests were used to compare the columnar and squamous epithelial groups, as well as individual sites and resulting p values are indicated on the graph.</p

Expression of gp340 in the ectocervix, endocervix, and colon.

<p>Tissue sections were stained by standard IHC for gp340 using the monoclonal antibody 143 IgG, as described in Materials and Methods. Representative images are shown. Brown stained areas are gp340 and cell nuclei appear blue. Sections of cervix and colon from two different subjects show diffuse granular staining of epithelial cell-associated gp340 throughout the ectocervical squamous epithelium <b>(A)</b>. The columnar epithelium of the endocervix <b>(B)</b> and colon <b>(C)</b> shows single-cell (Subject #1, Endocervix) and patchy (multiple adjacent cells, Subject #2, Endocervix) granular staining of the columnar epithelia in the endocervix and colon. Several examples of gp340⁺ cells are indicated with arrows. Negative controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132942#pone.0132942.g002" target="_blank">Fig 2A</a> (squamous epithelium) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132942#pone.0132942.s002" target="_blank">S2A Fig</a> (columnar epithelium).</p

Circumventing AKT-Associated Radioresistance in Oral Cancer by Novel Nanoparticle-Encapsulated Capivasertib

Background: Development of radioresistance in oral squamous cell carcinoma (OSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in recent decades. Effective strategies to overcome radioresistance are necessary to improve the therapeutic outcomes of radiotherapy in OSCC patients. Methods: Cells and xenograft tumors were irradiated using the Small Animal Radiation Research Platform. AKT inhibitor capivasertib (AZD5363) was encapsulated into cathepsin B-responsible nanoparticles (NPs) for tumor-specific delivery. Cell viability was measured by alamarBlue, cell growth was determined by colony formation and 3D culture, and apoptosis was assessed by flow cytometry with the staining of Fluorescein isothiocyanate (FITC) Annexin V and PI. An orthotopic tongue tumor model was used to evaluate the in vivo therapeutic effects. The molecular changes induced by the treatments were assessed by Western blotting and immunohistochemistry. Results: We show that upregulation of AKT signaling is the critical mechanism for radioresistance in OSCC cells, and AKT inactivation by a selective and potent AKT inhibitor capivasertib results in radiosensitivity. Moreover, relative to irradiation (IR) alone, IR combined with the delivery of capivasertib in association with tumor-seeking NPs greatly enhanced tumor cell repression in 3D cell cultures and OSCC tumor shrinkage in an orthotopic mouse model. Conclusions: These data indicate that capivasertib is a potent agent that sensitizes radioresistant OSCC cells to IR and is a promising strategy to overcome failure of radiotherapy in OSCC patients

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects