Pattern of DAP12 Expression in Leukocytes from Both Healthy and Systemic Lupus Erythematosus Patients

DAP12 is an ITAM-bearing transmembrane adaptor originally identified on the surface of Natural Killer cells. A broad expression among other immune cells was later found in myeloid and lymphoid cells. However, data on DAP12 expression pattern rely only on immunoblot and microarray analysis. Here, we describe the generation and the characterization of an anti-DAP12 monoclonal antibody. Using this novel reagent, we show that DAP12 expression is restricted to innate immune cells in basal condition. Since a decreased expression of DAP12 has been suggested in NK cells of systemic lupus erythematosus patients, we have further investigated the NK cell receptor repertoire and leukocyte expression of DAP12 in these patients and no major changes were detectable when compared to controls

Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65495–503/A*0201, BMLF1259–267/A*0201, or BZLF154–64/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection

The Eleventh and Twelfth Data Releases of the Sloan Digital Sky Survey: Final Data from SDSS-III

The third generation of the Sloan Digital Sky Survey (SDSS-III) took data from 2008 to 2014 using the original SDSS wide-field imager, the original and an upgraded multi-object fiber-fed optical spectrograph, a new near-infrared high-resolution spectrograph, and a novel optical interferometer. All of the data from SDSS-III are now made public. In particular, this paper describes Data Release 11 (DR11) including all data acquired through 2013 July, and Data Release 12 (DR12) adding data acquired through 2014 July (including all data included in previous data releases), marking the end of SDSS-III observing. Relative to our previous public release (DR10), DR12 adds one million new spectra of galaxies and quasars from the Baryon Oscillation Spectroscopic Survey (BOSS) over an additional 3000 deg2 of sky, more than triples the number of H-band spectra of stars as part of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE), and includes repeated accurate radial velocity measurements of 5500 stars from the Multi-object APO Radial Velocity Exoplanet Large-area Survey (MARVELS). The APOGEE outputs now include the measured abundances of 15 different elements for each star. In total, SDSS-III added 5200 deg2 of ugriz imaging; 155,520 spectra of 138,099 stars as part of the Sloan Exploration of Galactic Understanding and Evolution 2 (SEGUE-2) survey; 2,497,484 BOSS spectra of 1,372,737 galaxies, 294,512 quasars, and 247,216 stars over 9376 deg2; 618,080 APOGEE spectra of 156,593 stars; and 197,040 MARVELS spectra of 5513 stars. Since its first light in 1998, SDSS has imaged over 1/3 of the Celestial sphere in five bands and obtained over five million astronomical spectra. \ua9 2015. The American Astronomical Society

Analyse de la contribution des réponses lymphocytaires T spécifiques d'herpesvirus au répertoire alloréactif par criblage de lymphocytes T CD4+ ou CD8+ spécifiques du cytomégalovirus ou du virus d'Epstein-Barr

Les lymphocytes T exprimant des récepteurs pour l'antigène de type ab sont sélectionnés dans le thymus et ils sont de ce fait restreints à reconnaître des molécules du Complexe Majeur d'Histocompatibilité (CMH) du soi chargées en peptide. Néanmoins, les lymphocytes T sont fréquemment cross-réactifs vis à vis de molécules du CMH allogéniques. Bien que l'association entre des infections virales persistantes et le rejet d'allogreffes soit connue de longue date, peu d'exemples de cross-réactivités entre un complexe CMH du soi/peptide viral et des molécules du CMH allogéniques ont été décrits à ce jour. Nous avons criblé des populations de lymphocytes T CD4+ ou CD8+, dont la spécificité antigénique vis à vis de deux herpesvirus infectant une large partie de la population humaine, le virus d'Epstein-Barr et le cytomegalovirus, a été bien caractérisée afin d'évaluer la contribution de ces réponses au répertoire alloréactif. La plupart des lignées criblées ont été obtenues par tri immunomagnétique à l'aide de billes recouvertes de complexes CMH/peptide recombinants. Nos résultats montrent que des cellules T spécifiques d'épitopes d'herpesvirus montrent des réactions croisées avec des molécules du CMH allogéniques, manifestant une forte cytotoxicité vis à vis de différentes catégories de cellules cibles, et qu'elles pourraient ainsi contribuer au rejet d'allogreffes. Nous avons d'autre part mis en évidence que des lymphocytes T CD8+ peuvent manifester une alloréactivité médiée par un récepteur activateur de type NKR (récepteur de cellules Natural Killer) de la famille des LIR/ILT, renforçant la notion que des NKR activateurs contribuent à l'alloréactivitéT lymphocytes expressing ab T cell antigen receptors are selected in the thymus, and are thus restricted to recognize self Major Histocompatibility Complex (MHC) molecules loaded with peptides. Yet, T lymphocytes frequently cross-react with allogeneic MHC molecules. Though association between persistent viral infection and allograft rejection is well admitted, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactive potential of CD8+ or CD4+ T cell populations, specific for immunodominant epitopes derived from cytomegalovirus (HCMV) or Epstein-Barr virus (EBV), two herpesviruses that infect a large fraction of the human population. Most T cell lines that were screened were obtained by immunomagnetic sorting, using beads covered with recombinant peptide/MHC multimeric complexes. Our results show that herpesvirus-specific T cells frequently cross-react with allogeneic MHC molecules, exhibiting vigorous cytotoxicity toward different types of target cells. Thus, virus-specific T cells recognizing allo-MHC alleles may play an essential role in solid organ rejection. On the other hand, we bring evidence that CD8+ T lymphocytes can mediate alloresponses through an activating NKR receptor (Natural Killer cell receptor) of the LIR/ILT family, supporting the notion that activating NKR contribute to alloreactivityNANTES-BU Sciences (441092104) / SudocSudocFranceF

IL-21-Mediated Potentiation of Antitumor Cytolytic and Proinflammatory Responses of Human Vγ9Vδ2 T Cells for Adoptive Immunotherapy

International audienceVgamma9Vdelta2 T lymphocytes are a major human gammadelta T cell subset that react against a wide array of tumor cells, through recognition of phosphorylated isoprenoid pathway metabolites called phosphoantigens. Immunotherapeutic protocols targeting Vgamma9Vdelta2 T cells have yielded promising, yet limited, signs of antitumor efficacy. To improve these approaches, we analyzed the effects on gammadelta T cells of IL-21, a cytokine known to enhance proliferation and effector functions of CD8(+) T cells and NK cells. IL-21 induced limited division of phosphoantigen-stimulated Vgamma9Vdelta2 T cells, but did not modulate their sustained expansion induced by exogenous IL-2. Vgamma9Vdelta2 T cells expanded in the presence of IL-21 and IL-2 showed enhanced antitumor cytolytic responses, associated with increased expression of CD56 and several lytic molecules, and increased tumor-induced degranulation capacity. IL-21 plus IL-2-expanded Vgamma9Vdelta2 T cells expressed higher levels of inhibitory receptors (e.g., ILT2 and NKG2A) and lower levels of the costimulatory molecule NKG2D. Importantly, these changes were rapidly and reversibly induced after short-term culture with IL-21. Finally, IL-21 irreversibly enhanced the proinflammatory Th1 polarization of expanded Vgamma9Vdelta2 T cells when added at the beginning of the culture. These data suggest a new role played by IL-21 in the cytotoxic and Th1 programming of precommitted Ag-stimulated gammadelta T cells. On a more applied standpoint, IL-21 could be combined to IL-2 to enhance gammadelta T cell-mediated antitumor responses, and thus represents a promising way to optimize immunotherapies targeting this cell subset

Analysis of the TCR Vb repertoire of pp65_495–502/A*0201- or BZLF1/B*3501-sorted T cell lines, using TCR Vβ-specific mAb.

<p>The percentage of pp65/A*0201- or BZLF1/B*3501-specific T cells stained by the various anti-TCR Vβ mAb is mentioned according to the IMGT nomenclature (Beckman Coulter anti-TCR Vβ name is indicated in bracket). All T cell lines were derived from PBL, either from healthy donors or from RA patients.</p>a<p>Percentage determined by TCR sequencing (ref. 28). T cell lines exhibiting alloreactivity are marked in bold.</p

Enrichment in pp65_495–503/A*0201- or BZLF1_54–64/B*3501-specific T cells after sorting of CD8 T cells with pMHC magnetic multimers.

<p>(<b>A</b>) CD8 T cells, derived from the PBL from D01 and D03 were sorted with 245V mutated pp65_495–503/A*0201 multimers, then expanded in culture, and stained with PE-conjugated pp65_495–503/A*0201 tetramers and FITC-conjugated anti-CD3. (<b>B</b>) CD8 T cells derived from the PBL from D15 were sorted with 245V mutated BZLF1_54–64/B*3501 multimers, and then expanded in culture. Unsorted and sorted T cells were stained with PE-conjugated BZLF1/B35 tetramers and FITC-conjugated anti-CD3. The percentage of positive cells is indicated in the upper right quadrant.</p

Screening of CD8 T cell lines enriched in HCMV- or EBV-specific T cells for cross-reactivity to allogeneic MHC molecules.

a<p>CD8 T cell lines were screened on COS-7 cells transfected with individual HLA-encoding cDNA and TNF-α production was measured after a 6h-coculture. All T cell lines were PBL-derived. ND : not determined.</p

Screening of HCMV- or EBV-specific CD8 T cell lines for cross-recognition of allogeneic MHC molecules.

<p>CD8 T cell lines, sorted with recombinant pMHC multimeric complexes specific to HCMV (pp65_495–503/A*0201) or EBV lytic epitopes (BMLF1_259–267/A*0201 or BZLF1_54–64/B*3501), were tested: (A) for cytotoxicity toward a panel of 30 HLA-typed LCL, in a 4-h chromium release assay (Effector-Target ratio 15∶1). HLA-specific killing of LCL was observed for 3 of the 11 pp65/A2-specific T cell lines tested: T cell line from D01 killed A*3101⁺ LCL, T cell line from D03 killed A*3201⁺ LCL, and T cell line from D08 killed A*3001⁺ LCL. The BZLF1/B*3501-sorted T cell line from D15 killed LCL sharing Cw*0602 expression. Only T cell lines that showed alloresponse are displayed. LCL triggering an alloresponse are shown as well as some of the tested LCL which do not elicit a cytotoxic response. Cognate peptide-HLA complexes (pp65_495–503/A*0201 or BZLF1_54–64/B*3501) were used as positive control. Data are presented as the mean percentage lysis and are representative of 3 different experiments. (<b>B</b>) for TNF-α production toward COS-7 cells transfected with plasmids encoding class I HLA alleles. T cells were added 2 days after the transfection, and the TNF-α content of the supernatant, expressed in pg/mL, was estimated 6 h later by testing the toxicity of the supernatants for TNF-α sensitive WEHI-164 clone 13 cells. TNF-α production was observed for the pp65/A*0201-sorted T cell line from D03, that recognized selectively COS cells expressing HLA-A*3201, and for the BZLF1/B*3501 T cell line from D15 that recognized selectively COS cells expressing HLA-Cw*0602. Plasmids encoding class I HLA alleles that elicits a response are shown as well as some of the plasmid encoding class I HLA alleles that do not induce TNF-α secretion. T cell lines that did not produce TNF-α are not represented. Cognate peptide-HLA complexes (pp65_495–503/A*0201 or BZLF1_54–64/B*3501) were used as positive control. One out three independent experiments is shown.</p