The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy. Cancer Res; 71(5); 1597-606. Ó2011 AACR.

Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application.

The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.

Abstract:In the present study we provided a morphological and transcriptomic comparison of adult porcine adiposederived stem cells (ADSC) and bone marrow-derived stem cells (BMSC) as they differentiated in vitro towards the osteogenic and adipogenic lineages for up to 4 weeks. The long term goal of this comparison is to assess the possibility of using ADSC as a potential alternative to BMSC as a source of autologous adult stem cells in human therapies. Our data indicated that ADSC can differentiate into osteocytes and adipocytes similar to BMSC but with some differences. During the osteogenic differentiation both cell types went through morphological changes; however, while ADSC formed predominately osteogenic islands (nodules) in the culture dish, BMSC formed a continuous osteogenic sheet of small nodules. Transcriptomic analysis revealed that both cell types responded to the osteogenic induction. However, BGLAP mRNA expression did not increase in ADSC suggesting, together with the percentage area stained observed for Alizarin Red and von Kossa in ADSC, a lesser mineralization of bone matrix in this cell type compared to BMSC. During the adipogenic induction ADSC as well as BMSC were able to achieve the morphological and transcriptome changes characteristic of the adipogenic lineage. After 7 days of differentiation the expression patterns of DGAT2 and ADFP became greater in ADSC versus BMSC, which agreed with the larger lipid droplets formation observed in the ADSC by Oil Red O staining. Our findings represent an important step towards the assessment of using ADSC as an alternative to BMSC in therapeutic applications.

Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (␤ 2 -microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-Cspecific motif KYRV at residues 66 -76 of the ␣1-domain ␣-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locusspecific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.Class I human leukocyte antigens (called HLA) are cell-surface heterotrimers formed by a highly polymorphic heavy (44 kDa) chain, a non-polymorphic light (12 kDa) chain subunit (␤ 2 -microglobulin (␤ 2 m)3 ), and a short (8 -11-mer) peptide antigen derived from the degradation of intracellular proteins (1). The assembly pathway of most class I molecules involves an early interaction of the heavy chain, still free of ␤ 2 m, with calnexin, followed by association with ␤ 2 m and binding to the so-called peptide-loading complex. This is a supramolecular endoplasmic reticulum structure comprising, among others, two HLA-dedicated chaperones: TAP transporter associated with antigen processing) and the peptide editor/facilitator tapasin (1). Successful peptide loading results in tight association of the heavy chain with ␤ 2 m and the release of thermally stable, folded class I conformers (1-3). These are exported to the cell surface, where they activate and inhibit cytotoxic T lymphocytes expressing the rearranging T cell receptor and natural killer (NK) cells expressing non-rearranging receptors such as the killer immunoglobulin-like receptors, respectively (4).There are Ͼ1000 class I molecules, encoded by three highly polymorphic allelic series: HLA-A, -B, and -C (www. anthonynolan.org.UK/HIG/index.html). They share a conserved general architecture, a common peptide-loading pathway, and a similar set of functions, but also display a number of allel...

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), all of the MEM Abs unexpectedly reacted with ␤ 2 -microglobulin (␤ 2 m)-free and denatured (but not ␤ 2 m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas ␤ 2 m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to ␤ 2 m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, ␤ 2 m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.