CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor.

We found that sterile wounding of human skin induced epidermal expression of the antimicrobial (poly)peptides human beta-defensin-3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor through activation of the epidermal growth factor receptor. After skin wounding, the receptor was activated by heparin-binding epidermal growth factor that was released by a metalloprotease-dependent mechanism. Activation of the epidermal growth factor receptor generated antimicrobial concentrations of human beta-defensin-3 and increased the activity of organotypic epidermal cultures against Staphylococcus aureus. These data demonstrate that sterile wounding initiates an innate immune response that increases resistance to overt infection and microbial colonization.

Decreased clearance of Pseudomonas aeruginosa from airways of mice deficient in lysozyme M.

Lysozyme is a ubiquitous and abundant, cationic, antimicrobial polypeptide of leukocytes and epithelia, but its biological function in host defense is largely unexplored. To ascertain the role of lysozyme during bacterial infection of murine airways, we exposed the airways of lysozyme M-deficient (lys M-/-) mice to the pulmonary pathogen Pseudomonas aeruginosa and examined the host's response to infection. Despite partial compensation as a result of the appearance of lysozyme P in the infected airways of lys M-/- mice, these lys M-/- mice showed decreased clearance of P. aeruginosa compared with their lys M+/- or lys M+/+ littermates. Lysozyme contributes to optimal clearance of P. aeruginosa from the murine airways.

MicroRNA-related polymorphisms and non-Hodgkin lymphoma susceptibility in the Multicenter AIDS Cohort Study.

BACKGROUND: MicroRNAs, small non-coding RNAs involved in gene regulation, are implicated in lymphomagenesis. We evaluated whether genetic variations in microRNA coding regions, binding sites, or biogenesis genes (collectively referred to as miRNA-SNPs) were associated with risk of AIDS-associated non-Hodgkin lymphoma (AIDS-NHL), and serum levels of four lymphoma-related microRNAs. METHODS: Twenty-five miRNA-SNPs were genotyped in 180 AIDS-NHL cases and 529 HIV-infected matched controls from the Multicenter AIDS Cohort Study (MACS), and real-time polymerase chain reaction was used to quantify serum microRNA levels. Adjusted odds ratios (ORs) estimated using conditional logistic regression evaluated associations between miRNA-SNPs and AIDS-NHL risk. A semi-Bayes shrinkage approach was employed to reduce likelihood of false-positive associations. Adjusted mean ratios (MR) calculated using linear regression assessed associations between miRNA-SNPs and serum microRNA levels. RESULTS: DDX20 rs197412, a non-synonymous miRNA biogenesis gene SNP, was associated with AIDS-NHL risk (OR=1.34 per minor allele; 95% CI: 1.02-1.75), and higher miRNA-222 serum levels nearing statistical significance (MR=1.21 per minor allele; 95% CI: 0.98-1.49). MiRNA-196a2 rs11614913 was associated with decreased central nervous system (CNS) AIDS-NHL (CT vs. CC OR=0.52; 95% CI: 0.27-0.99). The minor allele of HIF1A rs2057482, which creates a miRNA-196a2 binding site, was associated with systemic AIDS-NHL risk (OR=1.73 per minor allele; 95% CI: 1.12-2.67), and decreased CNS AIDS-NHL risk (OR=0.49 per minor allele; 95% CI: 0.25-0.94). CONCLUSIONS: This study suggests that a few miRNA-SNPs are associated with AIDS-NHL risk and may modulate miRNA expression. These results support a role for miRNA in AIDS-NHL and may highlight pathways to be targeted for risk stratification or therapeutics.

Longitudinal analysis of peripheral blood T cell receptor diversity in patients with systemic lupus erythematosus by next-generation sequencing.

INTRODUCTION: T cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Clonal expansion of T cells correlating with disease activity has been observed in peripheral blood (PB) of SLE subjects. Recently, next-generation sequencing (NGS) of the T cell receptor (TCR) ß loci has emerged as a sensitive way to measure the T cell repertoire. In this study, we utilized NGS to assess whether changes in T cell repertoire diversity in PB of SLE patients correlate with or predict changes in disease activity. METHODS: Total RNA was isolated from the PB of 11 SLE patients. Each subject had three samples, collected at periods of clinical quiescence and at a flare. Twelve age-matched healthy controls (HC) were used for reference. NGS was used to profile the complementarity-determining region 3 (CDR3) of the rearranged TCR ß loci. RESULTS: Relative to the HC, SLE patients (at quiescence) demonstrated a 2.2-fold reduction in repertoire diversity in a given PB volume (P <0.0002), a more uneven distribution of the repertoire (Gini coefficient, HC vs SLE, P = 0.015), and a trend toward increased percentage of expanded clones in the repertoire (clone size >1.0%, HC vs SLE, P = 0.078). No significant correlation between the overall repertoire diversity and clinical disease activity was observed for most SLE patients with only two of eleven SLE patients showing a decreasing trend in repertoire diversity approaching the flare time point. We did not observe any overlap of CDR3 amino acid sequences or a preferential Vß or Jß gene usage among the top 100 expanded clones from all SLE patients. In both HC and SLE, the majority of the expanded clones were remarkably stable over time (HC = 5.5 ±0.5 months, SLE = 7.2 ±2.4 months). CONCLUSIONS: A significant decrease in T cell repertoire diversity was observed in PB of SLE patients compared to HC. However, in most SLE patients, repertoire diversity did not change significantly with increases in disease activity to a flare. Thus, without a priori knowledge of disease-specific clones, monitoring TCR repertoire in PB from SLE patients is not likely to be useful to predict changes in disease activity.

Serum microRNAs in HIV-infected individuals as pre-diagnosis biomarkers for AIDS-NHL.

OBJECTIVE: To determine if changes in levels of serum microRNAs (miRNAs) were seen preceding the diagnosis of AIDS-related non-Hodgkin lymphoma (AIDS-NHL). DESIGN: Serum miRNA levels were compared in 3 subject groups from the Multicenter AIDS Cohort Study: HIV-negative men (n = 43), HIV-positive men who did not develop NHL (n = 45), and HIV-positive men before AIDS-NHL diagnosis (n = 62, median time before diagnosis, 8.8 months). METHODS: A total of 175 serum-enriched miRNAs were initially screened to identify differentially expressed miRNAs among these groups and the results validated by quantitative polymerase chain reaction. Receiver-operating characteristic analysis was then performed to assess biomarker utility. RESULTS: Higher levels of miR-21 and miR-122, and a lower level of miR-223, were able to discriminate HIV-infected from the HIV-uninfected groups, suggesting that these miRNAs are biomarkers for HIV infection but are not AIDS-NHL specific. Among the HIV-infected groups, a higher level of miR-222 was able to discriminate diffuse large B-cell lymphoma (DLBCL) and primary central nervous system lymphoma (PCNSL) subjects from HIV-infected subjects who did not develop NHL, with area under the receiver-operating characteristic curve of 0.777 and 0.792, respectively. At miR-222 cutoff values of 0.105 for DLBCL and 0.109 for PCNSL, the sensitivity and specificity were 75% and 77%, and 80% and 82%, respectively. CONCLUSIONS: Altered serum levels of miR-21, miR-122, and miR-223 are seen in HIV-infected individuals. Higher serum level of miR-222 has clear potential as a serum biomarker for earlier detection of DLBCL and PCNSL among HIV-infected individuals.

B-cell activation induced microRNA-21 is elevated in circulating B cells preceding the diagnosis of AIDS-related non-Hodgkin lymphomas.

We show that microRNA-21 is significantly elevated in peripheral B cells of HIV-infected individuals who go on to develop AIDS-related non-Hodgkin lymphoma (n=13, <3 years prior to diagnosis) when compared with HIV-negative (n=18) or HIV-positive controls (n=21) (P<0.01). Moreover, miR-21 is overexpressed in activated B cells and can be induced by interleukin 4 alone, or with CD40 or immunoglobulin M co-stimulation, and lipopolysaccharides, suggesting that miR-21 may help maintain B-cell hyperactivation, contributing to lymphomagenesis.

Overexpression of microRNAs from the miR-17-92 paralog clusters in AIDS-related non-Hodgkin's lymphomas.

BACKGROUND: Individuals infected by HIV are at an increased risk for developing non-Hodgkin's lymphomas (AIDS-NHL). In the highly active antiretroviral therapy (HAART) era, there has been a significant decline in the incidence of AIDS-associated primary central nervous system lymphoma (PCNSL). However, only a modest decrease in incidence has been reported for other AIDS-NHL subtypes. Thus, AIDS-NHLs remain a significant cause of morbidity and mortality in HIV infected individuals. Recently, much attention has been directed toward the role of miRNAs in cancer, including NHL. Several miRNAs, including those encoded by the miR-17-92 polycistron, have been shown to play significant roles in B cell tumorigenesis. However, the role of miRNAs in NHL in the setting of HIV infection has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative realtime PCR to assess the expression of miRNAs from three different paralog clusters, miR-17-92, miR-106a-363, and miR-106b-25 in 24 cases of AIDS-NHLs representing four tumor types, Burkitt's lymphoma (BL, n = 6), diffuse large B-cell lymphoma (DLBCL, n = 8), primary central nervous system lymphoma (PCNSL, n = 5), and primary effusion lymphoma (PEL, n = 5). We also used microarray analysis to identify a differentiation specific miRNA signature of naïve, germinal center, and memory B cell subsets from tonsils (n = 4). miRNAs from the miR-17-92 paralog clusters were upregulated by B cells, specifically during the GC differentiation stage. We also found overexpression of these miRNA clusters in all four AIDS-NHL subtypes. Finally, we also show that select miRNAs from these clusters (miR-17, miR-106a, and miR-106b) inhibited p21 in AIDS-BL and DLBCL cases, thus providing a mechanistic role for these miRNAs in AIDS-NHL pathogenesis. CONCLUSION: Dysregulation of miR-17-92 paralog clusters is a common feature of AIDS-associated NHLs.

CD40 ligand (CD154) incorporated into HIV virions induces activation-induced cytidine deaminase (AID) expression in human B lymphocytes.

Most AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) arises from errors in immunoglobulin heavy-chain gene (IgH) class switch recombination (CSR) or somatic hypermutation (SHM), events that occur in germinal center (GC) B cells and require the activity of activation induced cytidine deaminase (AID). Several oncogenic viruses (EBV, HCV, HPV) can induce AID gene (AID) expression, and elevated AID expression is seen in circulating lymphocytes prior to AIDS-NHL diagnosis. Here, we report that HIV produced in peripheral blood mononuclear cells (PBMC) induced AID expression in normal human B cells. Since HIV produced in PBMC contains host cell CD40 ligand (CD40L) incorporated into the viral membrane, and CD40L is known to induce AID expression in human B cells, the role of virion-associated CD40L in HIV-induced AID expression was examined. Only viruses expressing functional CD40L were seen to induce AID expression; CD40L-negative HIV did not induce AID expression. The induction of AID expression by CD40L+ HIV was abrogated by addition of blocking anti-CD40L antibody. AID protein was detected in B cells exposed to CD40L+ HIV using intracellular multicolor flow cytometry, with most AID producing B cells expressing the CD71 activation marker on their surface. Therefore, HIV virions that express CD40L induce AID expression in B cells, and this induction appears to be due to a direct interaction between CD40L on these viruses and CD40 on B cells. These findings are consistent with a role for HIV in the direct stimulation of B cells, potentially leading to the accumulation of molecular lesions that have the potential to contribute to the development of NHL.

Soluble hemojuvelin is released by proprotein convertase-mediated cleavage at a conserved polybasic RNRR site.

As the principal iron-regulatory hormone, hepcidin plays an important role in systemic iron homeostasis. The regulation of hepcidin expression by iron loading appears to be unexpectedly complex and has attracted much interest. The GPI-linked membrane protein hemojuvelin (GPI-hemojuvelin) is an essential upstream regulator of hepcidin expression. A soluble form of hemojuvelin (s-hemojuvelin) exists in blood and acts as antagonist of GPI-hemojuvelin to downregulate hepcidin expression. The release of s-hemojuvelin is negatively regulated by both transferrin-bound iron (holo-Tf) and non-transferrin-bound iron (FAC), indicating s-hemojuvelin could be one of the mediators of hepcidin regulation by iron. In this report, we investigate the proteinase involved in the release of s-hemojuvelin and show that s-hemojuvelin is released by a proprotein convertase through the cleavage at a conserved polybasic RNRR site.

Differential regulation of beta-defensin expression in human skin by microbial stimuli.

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the beta-defensin family, human beta-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of beta-defensins is mediated by at least three different mechanisms.