Calcium Signaling in Dendritic Cells by Human or Mycobacterial Hsp70 Is Caused by Contamination and Is Not Required for Hsp70-mediated Enhancement of Cross-presentation

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation. Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation. Recent evidence has demonstrated that heat shock proteins (HSPs), 2The abbreviations used are: HSPheat shock protein familyAPCantigen-presenting cellCCRchemokine receptorCTLcytotoxic T lymphocyteDCdendritic cellHLAhuman leukocyte antigenHsp70stress-inducible member of the 70-kD familyLCLlymphoblastoid cell linemycmycobacterialrhurecombinant humanMART-1melanoma antigen recognized by T cells-1ncnucleotidepep70peptide sequence binding to Hsp70IFNinterferonLPSlipopolysaccharide. in addition to their roles as chaperones, can stimulate antigen-specific immune responses (1Calderwood S.K. Theriault J.R. Gong J. Eur. J. Immunol. 2005; 35: 2518-2527Crossref PubMed Scopus (130) Google Scholar). This immune stimulatory activity is thought to be activated after HSPs are released from cells either through necrosis or induced secretion (2Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1086) Google Scholar, 3Lancaster G.I. Febbraio M.A. J. Biol. Chem. 2005; 280: 23349-23355Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 4Lang A. Benke D. Eitner F. Engel D. Ehrlich S. Breloer M. Hamilton-Williams E. Specht S. Hoerauf A. Floege J. von Bonin A. Kurts C. J. Am. Soc. Nephrol. 2005; 16: 383-391Crossref PubMed Scopus (47) Google Scholar, 5Asea A. J. Biosci. 2007; 32: 579-584Crossref PubMed Scopus (77) Google Scholar). In their role as molecular chaperones, most HSPs exist in complexes with hydrophobic polypeptides. The contact of extracellular HSP:peptide complexes with dendritic cells (DCs) is thought to result in the efficient transfer of the chaperoned peptides into the antigen-presentation pathway of DCs allowing antigen-specific T cell activation. heat shock protein family antigen-presenting cell chemokine receptor cytotoxic T lymphocyte dendritic cell human leukocyte antigen stress-inducible member of the 70-kD family lymphoblastoid cell line mycobacterial recombinant human melanoma antigen recognized by T cells-1 nucleotide peptide sequence binding to Hsp70 interferon lipopolysaccharide. Mechanistically, HSP binding to surface receptors and HSP-induced signaling events are thought to be involved in antigen transfer to DCs and subsequent T cell stimulation. In this regard, a number of potential HSP receptors have been reported (6Becker T. Hartl F.U. Wieland F. J. Cell Biol. 2002; 158: 1277-1285Crossref PubMed Scopus (305) Google Scholar, 7Binder R.J. Vatner R. Srivastava P. Tissue Antigens. 2004; 64: 442-451Crossref PubMed Scopus (212) Google Scholar, 8Basu S. Binder R.J. Ramalingam T. Srivastava P.K. Immunity. 2001; 14: 303-313Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar, 9Calderwood S.K. Mambula S.S. Gray Jr., P.J. Theriault J.R. FEBS Lett. 2007; 581: 3689-3694Crossref PubMed Scopus (274) Google Scholar). Among these, CD40 and chemokine receptor 5 (CCR5) represent two interesting possibilities as these molecules are intricately linked to T cell and DC biology (10Kim C.H. Nagata K. Butcher E.C. J. Immunol. 2003; 171: 152-158Crossref PubMed Scopus (69) Google Scholar, 11Castellino F. Huang A.Y. Altan-Bonnet G. Stoll S. Scheinecker C. Germain R.N. Nature. 2006; 440: 890-895Crossref PubMed Scopus (610) Google Scholar). In this regard, CCR5 has been reported to act as a pattern recognition receptor for mycobacterial (myc)Hsp70 thus facilitating mycobacterial defense through mycHsp70-induced DC activation and subsequent T cell stimulation. The binding of mycHsp70 to CCR5, calcium signaling in myeloid DCs, phenotypic maturation, and cytokine secretion of DCs have been reported to underlie mycHsp70-assisted T cell activation (12Floto R.A. MacAry P.A. Boname J.M. Mien T.S. Kampmann B. Hair J.R. Huey O.S. Houben E.N. Pieters J. Day C. Oehlmann W. Singh M. Smith K.G. Lehner P.J. Science. 2006; 314: 454-458Crossref PubMed Scopus (130) Google Scholar, 13Whittall T. Wang Y. Younson J. Kelly C. Bergmeier L. Peters B. Singh M. Lehner T. Eur. J. Immunol. 2006; 36: 2304-2314Crossref PubMed Scopus (54) Google Scholar). We have previously demonstrated that CCR5 can mediate unique signaling events (14Mack M. Kleinschmidt A. Bruhl H. Klier C. Nelson P.J. Cihak J. Plachy J. Stangassinger M. Erfle V. Schlondorff D. Nat. Med. 2000; 6: 769-775Crossref PubMed Scopus (466) Google Scholar, 15Weber C. Weber K.S. Klier C. Gu S. Wank R. Horuk R. Nelson P.J. Blood. 2001; 97: 1144-1146Crossref PubMed Scopus (210) Google Scholar). In addition, we have shown that human Hsp70 from melanoma cells can chaperone tumor-associated peptides (tyrosinase) and deliver them to DCs allowing cross-presentation and T cell stimulation (16Noessner E. Gastpar R. Milani V. Brandl A. Hutzler P.J. Kuppner M.C. Roos M. Kremmer E. Asea A. Calderwood S.K. Issels R.D. J. Immunol. 2002; 169: 5424-5432Crossref PubMed Scopus (220) Google Scholar). Recently, we used recombinant human (rhu)Hsp70 to define in more detail the critical parameters for the induction of immune effector responses (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Highly purified and endotoxin-depleted rhuHsp70 was found to efficiently deliver exogenous peptides to DCs resulting in T cell activation, provided that peptide and Hsp70 had formed stable complexes before exposure to DCs. Phenotypic maturation or cytokine secretion of DCs was not observed and not required for the rhuHsp70-mediated enhancement of cross-presentation and T cell stimulation. Here we used highly purified rhuHsp70 and mycHsp70 proteins and examined whether mycHsp70 also enhances cross-presentation of exogenous antigenic protein sequences and tested whether calcium signaling or CCR5 were required for Hsp70-assisted antigen presentation and T cell stimulation. Reagents and Peptides—ADP, ionomycin, suramin, Escherichia coli-derived LPS (strain 0111:B4), and reagents for calcium signaling (Indo-I AM, Pluronic F-127, DMSO) were purchased from Sigma. All buffers and solutions were prepared using aqua ad injectabilia (Braun Melsungen, Germany). Peptides were purchased from Biosyntan GmbH (Berlin, Germany). The peptides were synthesized as hybrids of the nonameric HLA-A2 binding motif with the N-terminal octameric sequence HWDFAWPW (here named pep70) (pep70-MART-1: GSGHWDFAWPWGSGLAGIGILTV). Recombinant Hsp70 Proteins—Recombinant human (rhu)-Hsp70 protein was either purchased from Stressgen, Victoria, BC, Canada (ESP-555) or was prepared in our own laboratory as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). After ADP affinity chromatography, buffer was exchanged using PD10 columns followed by extensive dialysis (48 h, 5000× buffer volume) and diafiltration (Vivaspin 20, 30,000 MWCO PES, Sartorius Stedim Biotech). To assess the influence of nucleotide carryover, rhuHsp70 preparations were used after PD10 column and diafiltration with a less intensive dialysis step (Hsp70/nc+). Endotoxin was depleted, as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Only proteins with endotoxin below 0.5 EU/mg rhuHsp70 were used. Full-length mycHsp70 was obtained from Lionex Diagnostics & Therapeutics GmbH, Braunschweig, Germany (Product: M. tuberculosis Hsp70, LRP 0003.3; batch: 07-1 lot2). The cDNA coding for the C-terminal substrate binding domain of mycHsp70, mycHsp70-(359–625), kindly provided by P. J. Lehner, Cambridge (UK), was subcloned into the vector pET21d (Novagen/EMD Chemicals, Gibbstown, NJ). C-terminally His6-tagged recombinant protein was produced in E. coli strain Rosetta2(DE3) (Invitrogen, Karlsruhe, Germany) and purified using Ni-nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) as described (18Udono H. Saito T. Ogawa M. Yui Y. Methods. 2004; 32: 21-24Crossref PubMed Scopus (14) Google Scholar). Endotoxin depletion was performed as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). APCs and Cell Cultures—DCs were differentiated from monocytes of PBMC of healthy donors (CD14+ magnetic bead selection, Miltenyi) using IL4/GM-CSF exactly as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Cells were used without further maturation (i.e. non-matured IL4/GM-CSF-differentiated myeloid DCs). The local ethics commission approved these studies, and donors gave informed consent. Human B-lymphoblastoid B cell line (B-LCL L721.45) and CTL A42-MART (HLA-A2-restricted Melan-A/MART-1-peptide (LAGIGILTV)-specific) were cultured as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Cross-presentation—Cross-presentation was performed exactly as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Briefly, in parallel wells of a 96-well cell culture plate identical amounts of pep70-MART-peptide (70 nm) alone or together with rhuHsp70 (1 μm) or mycHsp70-(359–625) (1 μm) were incubated in HKM buffer (total volume of 30 μl) for 4 h at room temperature for complex formation. B-LCL 721.45 and CTL A42-MART were added resulting in a 1:7 dilution of rhuHsp70 and peptide. The concentrations declared in text/figure correspond to the final concentrations in the APC/T cell mixture. After 24 h at 37 °C, supernatants were harvested, and IFN-γ was measured by ELISA (BD OptEIA™, BD Biosciences Pharmingen). Control samples, containing all components except the peptide, were used to determine IFN-γ background, which was subtracted from the experimental samples. Flow Cytometry—Surface expression of molecules was determined by flow cytometry. 0.1 × 106 cells (DCs or B-LCL) after a 48-h incubation with 300 nm rhuHsp70, LPS (1 μg/ml) or without stimulus, were with CD40 BD Biosciences or (BD in containing After 30 of incubation cells were and with the and Cell myeloid DCs were incubated with 300 nm rhuHsp70, LPS (1 or without for h at 37 in the supernatants were measured using the and concentrations were using the was performed as and in calcium were measured with using (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). ADP mycHsp70-(359–625) (1 rhuHsp70 mycHsp70 Lionex (1 or the solutions to the 300 nm rhuHsp70 or mycHsp70 protein were added after the cells were measured without stimulation for The solutions were obtained by protein through of the through a with molecular (Vivaspin 20, MWCO DCs were with for 5 the buffers without were measured and not result in a calcium in DCs by of rhuHsp70 and mycHsp70 (12Floto R.A. MacAry P.A. Boname J.M. Mien T.S. Kampmann B. Hair J.R. Huey O.S. Houben E.N. Pieters J. Day C. Oehlmann W. Singh M. Smith K.G. Lehner P.J. Science. 2006; 314: 454-458Crossref PubMed Scopus (130) Google Scholar, 13Whittall T. Wang Y. Younson J. Kelly C. Bergmeier L. Peters B. Singh M. Lehner T. Eur. J. Immunol. 2006; 36: 2304-2314Crossref PubMed Scopus (54) Google Scholar, P.A. B. R.A. Smith K.G. Oehlmann W. Singh M. Lehner P.J. Immunity. 2004; Full Text Full Text PDF PubMed Scopus Google have that mycHsp70 a calcium in DCs resulting in DC maturation and antigen-specific T cell activation. results using highly purified rhuHsp70 had shown that rhuHsp70 enhances cross-presentation and T cell stimulation without the DC (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). We and our rhuHsp70 to the mycHsp70 used in the (12Floto R.A. MacAry P.A. Boname J.M. Mien T.S. Kampmann B. Hair J.R. Huey O.S. Houben E.N. Pieters J. Day C. Oehlmann W. Singh M. Smith K.G. Lehner P.J. Science. 2006; 314: 454-458Crossref PubMed Scopus (130) Google Scholar, 13Whittall T. Wang Y. Younson J. Kelly C. Bergmeier L. Peters B. Singh M. Lehner T. Eur. J. Immunol. 2006; 36: 2304-2314Crossref PubMed Scopus (54) Google Scholar, P.A. B. R.A. Smith K.G. Oehlmann W. Singh M. Lehner P.J. Immunity. 2004; Full Text Full Text PDF PubMed Scopus Google for to calcium signals in DCs. Hsp70 and in E. are purified using affinity chromatography, and buffer A. H. M. A. B. Nat. Biol. PubMed Scopus Google Scholar). are of calcium signals in immune cells A. D. S. A. F. G. J. Immunol. 2001; PubMed Scopus Google Scholar, F. D. J.M. J. Biol. 2004; PubMed Scopus Google Scholar, M. T. A. M. J. E. Blood. 2005; PubMed Scopus Google Scholar). we that or ADP have caused the reported calcium To for by in the protein we prepared a by the mycHsp70 protein from the protein by through a with a and that the had Hsp70 protein or protein the result for results were seen for not for calcium signaling we observed that the mycHsp70 induced a calcium in DCs that was to that seen with the mycHsp70 protein We used which had a less dialysis after affinity chromatography, as a human Hsp70 less induced a calcium in to the mycHsp70 the rhuHsp70 induced a We the rhuHsp70 protein from the less protein as performed for mycHsp70 and tested the for to a calcium in DCs. was found for the mycHsp70 the of rhuHsp70 identical calcium responses as seen with the line we tested solutions of ADP and observed calcium signals nm ADP which to the of nucleotide in our less gave identical line nm the calcium of DCs was not in nm the of the to 300 nm rhuHsp70 was not that with the that nucleotides that Hsp70 were for the calcium with or of the rhuHsp70 protein with this we found that the induced by this less rhuHsp70 protein was by the suramin, as was the to the ADP To the that the mycHsp70 and rhuHsp70 proteins not calcium signals in DCs, we used of rhuHsp70, and the C-terminal substrate binding domain of mycHsp70, mycHsp70-(359–625) previously described as activity with to calcium signaling, antigen and T cell activation P.A. B. R.A. Smith K.G. Oehlmann W. Singh M. Lehner P.J. Immunity. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). this protein the binding domain and is not purified the rhuHsp70 the mycHsp70-(359–625) induced a calcium in DCs calcium signals have been reported to be through CCR5 we also measured the of surface CCR5 expression of our DCs by cell shown in the DCs used in these were the that of CCR5 have caused the to a calcium we surface expression and cytokine secretion of DCs before and after incubation with rhuHsp70 preparations were was strongly and of nucleotides calcium signaling the was less and nucleotides signaling All Hsp70 preparations were endotoxin-depleted and induced in DC or cytokine secretion H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). LPS was used as a shown in the rhuHsp70 preparations not surface or whether nucleotides and had calcium signaling activity or were highly purified and not calcium LPS these neither the the rhuHsp70 in endotoxin-depleted the cytokine or chemokine of DCs, LPS induced the secretion these and are shown as were also not These results that concentrations of as present in less rhuHsp70 which calcium signals in DCs, not phenotypic or cytokine surface and secretion can be induced by LPS with signaling activity in H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). We not these with mycHsp70, the of mycHsp70 is reported to be by J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google which is to phenotypic and cytokine and chemokine secretion through F. Smith A. J. Immunol. 2003; PubMed Scopus Google Scholar). we the to or from protein preparations have been our to from of potential Human and Hsp70 without or binding and calcium signaling were reported to be linked to the T cell stimulatory activity of mycHsp70 (12Floto R.A. MacAry P.A. Boname J.M. Mien T.S. Kampmann B. Hair J.R. Huey O.S. Houben E.N. Pieters J. Day C. Oehlmann W. Singh M. Smith K.G. Lehner P.J. Science. 2006; 314: 454-458Crossref PubMed Scopus (130) Google Scholar, 13Whittall T. Wang Y. Younson J. Kelly C. Bergmeier L. Peters B. Singh M. Lehner T. Eur. J. Immunol. 2006; 36: 2304-2314Crossref PubMed Scopus (54) Google Scholar). that the calcium signals of mycHsp70 or were caused by nucleotide of the protein we whether in the of calcium was to antigen cross-presentation and activation. To this we tested the of in our cross-presentation (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google and to T cells to the The of the are highly purified Hsp70 the antigenic peptide and the antigen-specific APCs were incubated in parallel with identical concentrations of peptide either with or without the addition of and Hsp70 are incubated to complex formation. The of peptide cross-presentation was by the of APCs to IFN-γ in antigen-specific CTL as described (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). APCs we the B cell line to in parallel the of CCR5 expression. shown in a IFN-γ was obtained peptide was to the APCs as a complex with either or These results that mycHsp70 enhances antigen cross-presentation to rhuHsp70, and neither rhuHsp70 mycHsp70 CCR5 or calcium for this Here we that highly purified rhuHsp70 protein and the C-terminal mycHsp70 not calcium signals in APCs require CCR5 or calcium signals to enhance exogenous antigen cross-presentation for T cell stimulation. we found that nucleotides and to be recombinant human and mycobacterial Hsp70 preparations of calcium signals in myeloid DCs. reported previously (17Bendz H. Ruhland S.C. Pandya M.J. Hainzl O. Riegelsberger S. Brauchle C. Mayer M.P. Buchner J. Issels R.D. Noessner E. J. Biol. Chem. 2007; 282: 31688-31702Abstract Full Text Full Text PDF PubMed Scopus (109) Google the activity to stimulate antigen-specific T cell responses is the of Hsp70 to complexes with the exogenous peptide without DC phenotypic or The observed of calcium signaling further the that the of and to immune responses be of their activity as molecular and is to to Hsp70 proteins to this was by H. D. D. Immunol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Immunol. 2004; PubMed Scopus Google Scholar, J. Immunol. 2005; PubMed Scopus Google Scholar). were the of Hsp70 and HSP preparations H. D. S. S. C. H. D. Eur. J. Immunol. 2002; 32: PubMed Scopus Google Scholar, A. von Bonin A. R. Immunol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. 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