CD160 isoforms and regulation of CD4 and CD8 T-cell responses.

BACKGROUND: Coexpression of CD160 and PD-1 on HIV-specific CD8+ T-cells defines a highly exhausted T-cell subset. CD160 binds to Herpes Virus Entry Mediator (HVEM) and blocking this interaction with HVEM antibodies reverses T-cell exhaustion. As HVEM binds both inhibitory and activatory receptors, our aim in the current study was to assess the impact of CD160-specific antibodies on the enhancement of T-cell activation. METHODS: Expression of the two CD160 isoforms; glycosylphosphatidylinositol-anchored (CD160-GPI) and the transmembrane isoforms (CD160-TM) was assessed in CD4 and CD8 primary T-cells by quantitative RT-PCR and Flow-cytometry. Binding of these isoforms to HVEM ligand and the differential capacities of CD160 and HVEM specific antibodies to inhibit this binding were further evaluated using a Time-Resolved Fluorescence assay (TRF). The impact of both CD160 and HVEM specific antibodies on enhancing T-cell functionality upon antigenic stimulation was performed in comparative ex vivo studies using primary cells from HIV-infected subjects stimulated with HIV antigens in the presence or absence of blocking antibodies to the key inhibitory receptor PD-1. RESULTS: We first show that both CD160 isoforms, CD160-GPI and CD160-TM, were expressed in human primary CD4+ and CD8+ T-cells. The two isoforms were also recognized by the HVEM ligand, although this binding was less pronounced with the CD160-TM isoform. Mechanistic studies revealed that although HVEM specific antibodies blocked its binding to CD160-GPI, surprisingly, these antibodies enhanced HVEM binding to CD160-TM, suggesting that potential antibody-mediated HVEM multimerization and/or induced conformational changes may be required for optimal CD160-TM binding. Triggering of CD160-GPI over-expressed on Jurkat cells with either bead-bound HVEM-Fc or anti-CD160 monoclonal antibodies enhanced cell activation, consistent with a positive co-stimulatory role for CD160-GPI. However, CD160-TM did not respond to this stimulation, likely due to the lack of optimal HVEM binding. Finally, ex vivo assays using PBMCs from HIV viremic subjects showed that the use of CD160-GPI-specific antibodies combined with blockade of PD-1 synergistically enhanced the proliferation of HIV-1 specific CD8+ T-cells upon antigenic stimulation. CONCLUSIONS: Antibodies targeting CD160-GPI complement the blockade of PD-1 to enhance HIV-specific T-cell responses and warrant further investigation in the development of novel immunotherapeutic approaches.

sTREM2 Differentially Affects Cytokine Expression in Myeloid-Derived Cell Models via MAPK-JNK Signaling Pathway.

TREM2 is a critical innate immune receptor primarily expressed on myeloid-derived cells, such as microglia and macrophages. Mutations in TREM2 are linked to several neurodegenerative diseases including Alzheimer's disease (AD). TREM2 can be cleaved from the cell membrane and released as soluble TREM2 (sTREM2). sTREM2 levels are shown to peak prior to AD, with its levels fluctuating throughout disease progression. However, the mechanism by which sTREM2 may affect innate immune responses is largely uncharacterized. In this study, we investigated whether sTREM2 can induce inflammatory response in myeloid-derived THP-1 monocytes and macrophages and characterized the signaling mechanisms involved. Our results show that sTREM2 was capable of stimulating the expression of several inflammatory cytokines in THP-1 cells throughout the time course of 2 h to 8 h but inducing anti-inflammatory cytokine expression at later time points. A TREM2 antibody was capable of inhibiting the expression of some cytokines induced by sTREM2 but enhancing others. The complex of sTREM2/TREM2 antibody was shown to enhance IL-1ß expression, which was partially blocked by an NLRP3 specific inhibitor, indicating that the complex activated the NRLP3 inflammasome pathway. sTREM2 was also shown to have differential effects on cytokine expression in M0, M1, and M2 macrophages differentiated from THP-1 cells. sTREM2 has a more stimulating effect on cytokine expression in M0 macrophages, less of an effect on M2 macrophages, and some inhibitory effects on cytokine expression in M1 macrophages at early time points. Analyses of several signaling pathways revealed that sTREM2-induced expression of cytokines occurs mainly through MAPK-JNK signaling. Our work reveals differential effects of sTREM2 on cytokine expression profiles of THP-1 cells and macrophages and demonstrates that the MAPK-JNK signaling pathway is mainly responsible for sTREM2-induced cytokine expression.

Nucleotide competing reverse transcriptase inhibitors: discovery of a series of non-basic benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

A HTS screen led to the identification of a benzofurano[3,2-d]pyrimidin-2-one core structure which upon further optimization resulted in 1 as a potent HIV-1 nucleotide competing reverse transcriptase inhibitor (NcRTI). Investigation of the SAR at N-1 allowed significant improvements in potency and when combined with the incorporation of heterocycles at C-8 resulted in potent analogues not requiring a basic amine to achieve antiviral activity. Additional modifications at N-1 resulted in 33 which demonstrated excellent antiviral potency and improved physicochemical properties.

Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors.

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells.

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the 'secretome'). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the 'signal trap' strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.

Identification of a conserved cluster of skin-specific genes encoding secreted proteins.

Terminal differentiation of keratinocytes results in the formation of a cornified layer composed of cross-linked intracellular and extracellular material. Using a signal trap expression screening strategy, we have identified four cDNAs encoding secreted proteins potentially involved in this process. One of the cDNAs is identical to the short isoform of suprabasin, a recently described epidermis-specific protein, which is shown here to contain a functional secretory signal. The second cDNA, sk89, encodes a protein of 493 amino acids, rich in glycine and serine residues. The third cDNA encodes a C-terminal fragment of SK89 (amino acids 410-493). It comprises exons 13 to 18 of the sk89 locus but transcription starts at an isoform-specific exon encoding a distinct secretory signal. The fourth cDNA encodes keratinocyte differentiation-associated protein (KDAP), a precursor protein of 102 amino acids. Subcellular localization by immunofluorescence and detection of the tagged proteins by Western blotting confirmed that the four proteins are secreted. Northern analysis and in situ hybridization revealed that expression of the corresponding genes was restricted to the suprabasal keratinocytes of the epidermis. These genes encoding epidermis-specific secreted products are found in a conserved cluster on human chromosome 19q13.12 and on mouse chromosome 7A3.

Bril: a novel bone-specific modulator of mineralization.

In the course of attempting to define the bone "secretome" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Bril overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype.

Although a number of secreted factors have been demonstrated to be bone regulators, none of these are unique to bone. Using a viral-based signal-trap strategy we have identified a novel gene we have termed "osteocrin." A 1280-bp mRNA encodes osteocrin producing a mature protein of 103 amino acids with a molecular mass of 11.4 kDa. Osteocrin shows no homology with any known gene except for two conserved sequence motifs reminiscent of dibasic cleavage sites found in peptide hormone precursors. Immunofluorescence and Western blot analysis confirmed the secretory nature of osteocrin. Two protein species were identified in the medium of cells overexpressing osteocrin, a full-length 11.4 kDa species and a processed approximately 5 kDa species. Mutation of the 76KKKR79 dibasic cleavage site abolished the appearance of this smaller osteocrin fragment. By in situ hybridization in mouse embryos, osteocrin was expressed specifically in Cbfa-1-positive, osteocalcin-negative osteoblasts. Immunohistochemistry on adult mouse bone showed osteocrin localization in osteoblasts and young osteocytes. By Northern blot analysis, osteocrin expression was only detected in bone, expression peaking just after birth and decreasing markedly with age. In primary osteoblastic cell cultures osteocrin expression coincided with matrix formation then decreased in very mature cultures. Treatment of cultures with 1,25-dihydroxyvitamin D3 resulted in a rapid dose-dependent down-regulation of osteocrin expression, suggesting direct regulation. Chronic treatment of primary cultures with osteocrin-conditioned media inhibited mineralization and reduced osteocalcin and alkaline phosphatase expression. These results suggest that osteocrin represents a novel, unique vitamin D-regulated bone-specific protein that appears to act as a soluble osteoblast regulator.