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Tumor Necrosis Factor-α and CD80 Modulate CD28 Expression through a Similar Mechanism of T-cell Receptor-independent Inhibition of Transcription

2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês

10.1074/jbc.m402194200

ISSN

1083-351X

Autores

Dorothy E. Lewis, Maria Merched-Sauvage, Jörg J. Goronzy, Cornelia M. Weyand, Abbe N. Vallejo,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

Replicative senescence of human T cells is characterized by the loss of CD28 expression, exemplified by the clonal expansion of CD28null T cells during repeated stimulation in vitro as well as in chronic inflammatory and infectious diseases and in the normal course of aging. Because CD28 is the major costimulatory receptor for the induction of T cell-mediated immunity, the mechanism(s) underlying CD28 loss is of paramount interest. Current models of replicative senescence involve protracted procedures to generate CD28null cells from CD28+ precursors; hence, a T-cell line model was used to examine the dynamics of CD28 expression. Here, we show the versatility of the JT and Jtag cell lines in tracking CD28null ↔ CD28hi phenotypic transitions. JT and Jtag cells were CD28null and CD28lo, respectively, but expressed high levels of CD28 when exposed to phorbol 12-myristate 13-acetate. This was a result of the reconstitution of the CD28 gene transcriptional initiator (INR). Tumor necrosis factor-α reduced CD28 expression because of the inhibition of INR-driven transcription. Ligation of CD28 by an antibody or by CD80 also down-regulated CD28 transcription through the same mechanism, providing evidence that CD28 can generate a T cell receptor-independent signal with a unique biological outcome. Collectively, these data unequivocally demonstrate the critical role of the INR in the regulation of CD28 expression. T cell lines with transient expression of CD28 are invaluable in the dissection of the biochemical processes involved in the transactivation of the CD28 INR, the silencing of which is a key event in the ontogenesis of senescent T cells. Replicative senescence of human T cells is characterized by the loss of CD28 expression, exemplified by the clonal expansion of CD28null T cells during repeated stimulation in vitro as well as in chronic inflammatory and infectious diseases and in the normal course of aging. Because CD28 is the major costimulatory receptor for the induction of T cell-mediated immunity, the mechanism(s) underlying CD28 loss is of paramount interest. Current models of replicative senescence involve protracted procedures to generate CD28null cells from CD28+ precursors; hence, a T-cell line model was used to examine the dynamics of CD28 expression. Here, we show the versatility of the JT and Jtag cell lines in tracking CD28null ↔ CD28hi phenotypic transitions. JT and Jtag cells were CD28null and CD28lo, respectively, but expressed high levels of CD28 when exposed to phorbol 12-myristate 13-acetate. This was a result of the reconstitution of the CD28 gene transcriptional initiator (INR). Tumor necrosis factor-α reduced CD28 expression because of the inhibition of INR-driven transcription. Ligation of CD28 by an antibody or by CD80 also down-regulated CD28 transcription through the same mechanism, providing evidence that CD28 can generate a T cell receptor-independent signal with a unique biological outcome. Collectively, these data unequivocally demonstrate the critical role of the INR in the regulation of CD28 expression. T cell lines with transient expression of CD28 are invaluable in the dissection of the biochemical processes involved in the transactivation of the CD28 INR, the silencing of which is a key event in the ontogenesis of senescent T cells. The CD28 molecule is a membrane glycoprotein with nearly restricted expression to T lymphocytes. Its role as the major costimulatory receptor for the induction and maintenance of T-cell activation and differentiation of effector function is well documented (1Chambers C.A. Allison J.P. Curr. Opin. Cell Biol. 1999; 11: 203-210Crossref PubMed Scopus (350) Google Scholar). In humans, chronological aging is associated with the in vivo accumulation of T cells that are deficient in CD28 expression (2Posnett D.N. Sinha R. Kabak S. Russo C. J. Exp. Med. 1994; 179: 609-618Crossref PubMed Scopus (719) Google Scholar, 3Vallejo A.N. Nestel A.R. Schirmer M. Weyand C.M. Goronzy J.J. J. Biol. Chem. 1998; 273: 8119-8129Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Although mechanisms leading to immunosenescence are complex, CD28null T cells are the most consistent biological indicators of aging in the immune system. Indeed, clinical studies have shown that a high frequency of the CD28null T cells is a key predictor of immunoincompetence in the elderly (4Goronzy J.J. Fulbright J.W. Crowson C.S. Poland G.A. O'Fallon W.M. Weyand C.M. J. Virol. 2001; 75: 12182-12187Crossref PubMed Scopus (358) Google Scholar, 5Saurwein-Teissl M. Lung T.L. Marx F. Gschosser C. Asch E. Blasko I. Parson V. Bock V. Schonitzer V. Trannoy V. Grubeck-Loebenstein B. J. Immunol. 2002; 168: 5893-5899Crossref PubMed Scopus (422) Google Scholar). These unusual T cells have also been found among patients with various inflammatory syndromes (6Neil G.A. Summers R.W. Cheyne B.A. Carpenter C. Huang W.L. Waldschmidt T.J. Dig. Dis. Sci. 1994; 39: 1900-1908Crossref PubMed Scopus (17) Google Scholar, 7Martens P.B. Goronzy J.J. Schaid D.J. Weyand C.M. Arthritis Rheum. 1997; 40: 1106-1114Crossref PubMed Scopus (262) Google Scholar, 8Moosig F. Csernok E. Wang G. Gross W.L. Clin. Exp. Immunol. 1998; 114: 113-118Crossref PubMed Scopus (137) Google Scholar, 9Honda M. Mengesha E. Albano S. Nichols W.S. Wallace D.J. Metzger A. Klinenberg J.R. Linker-Israeli M. Clin. Immunol. 2001; 99: 211-221Crossref PubMed Google Scholar, 10Schirmer M. Goldberger C. Wurzner R. Duftner C. Pfeiffer K.P. Clausen J. Neumayr G. Falkenbach A. Arthritis Res. 2002; 4: 71-76Crossref PubMed Scopus (96) Google Scholar) and chronic infections, including HIV 1The abbreviations used are: HIV, human immunodeficiency virus; INR, initiator; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcription; TNF, tumor necrosis factor; RPA, RNase protection assay; EMSA, electrophoretic mobility shift assay; TdT, terminal deoxyribonucleotidyl transferase; RPA, RNase protection assay; TdT, terminal deoxyribonucleotidyl transferase; TCR, T cell receptor. (11Dutra W.O. Martins-Filho O.A. Cancado J.R. Pinto-Dias J.C. Brener Z. Gazzinelli G. Carvalho J.F. Colley D.G. Scand. J. Immunol. 1996; 43: 88-93Crossref PubMed Scopus (80) Google Scholar, 12Schlienger K. Uyemura K. Jullien D. Sieling P.A. Rea T.H. Peter S. Modlin R.L. J. Immunol. 1998; 161: 2407-2413PubMed Google Scholar, 13Lewis D.E. Yang L. Luo W. Wang X. Rodgers J.R. AIDS. 1999; 13: 1029-1033Crossref PubMed Scopus (24) Google Scholar, 14Appay V. Dunbar P.R. Callan M. Klenerman P. Gillespie G.M. Papagno L. Ogg G.S. King A. Lechner F. Spina C.A. Little S. Havlir D.V. Richman D.D. Gruener N. Pape G. Waters A. Easterbrook P. Salio M. Cerundolo V. McMichael A.J. Rowland-Jones S.L. Nat. Med. 2002; 8: 379-385Crossref PubMed Scopus (1332) Google Scholar). In these pathological conditions, CD28null T cells are postulated to represent prematurely senescent T cells as a result of persistent immune activation (15Vallejo A.N. Weyand C.M. Goronzy J.J. Trends Mol. Med. 2004; 10: 119-124Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The notion that CD28null T cells have advanced senescent features comes from observations that they are highly oligoclonal (2Posnett D.N. Sinha R. Kabak S. Russo C. J. Exp. Med. 1994; 179: 609-618Crossref PubMed Scopus (719) Google Scholar, 5Saurwein-Teissl M. Lung T.L. Marx F. Gschosser C. Asch E. Blasko I. Parson V. Bock V. Schonitzer V. Trannoy V. Grubeck-Loebenstein B. J. Immunol. 2002; 168: 5893-5899Crossref PubMed Scopus (422) Google Scholar, 16Schmidt D. Martens P.B. Weyand C.M. Goronzy J.J. Mol. Med. 1996; 2: 608-618Crossref PubMed Google Scholar) and have significantly shortened telomeres compared with their CD28+ counterparts (9Honda M. Mengesha E. Albano S. Nichols W.S. Wallace D.J. Metzger A. Klinenberg J.R. Linker-Israeli M. Clin. Immunol. 2001; 99: 211-221Crossref PubMed Google Scholar, 17Monteiro J. Batliwalla F. Ostrer H. Gregersen P.K. J. Immunol. 1996; 156: 3587-3590PubMed Google Scholar). Therefore, they have extremely limited proliferative potential (18Spaulding C. Guo W. Effros R.B. Exp. Gerontol. 1999; 34: 633-644Crossref PubMed Scopus (210) Google Scholar, 19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar). However, they are long-lived cells because of their resistance to apoptosis (18Spaulding C. Guo W. Effros R.B. Exp. Gerontol. 1999; 34: 633-644Crossref PubMed Scopus (210) Google Scholar, 20Vallejo A.N. Schirmer M. Weyand C.M. Goronzy J.J. J. Immunol. 2000; 165: 6301-6307Crossref PubMed Scopus (146) Google Scholar). The CD4+ T-cell compartment of elderly persons and patients with inflammatory conditions or chronic infections may consist of up to 50% CD28null T cells (3Vallejo A.N. Nestel A.R. Schirmer M. Weyand C.M. Goronzy J.J. J. Biol. Chem. 1998; 273: 8119-8129Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 7Martens P.B. Goronzy J.J. Schaid D.J. Weyand C.M. Arthritis Rheum. 1997; 40: 1106-1114Crossref PubMed Scopus (262) Google Scholar, 11Dutra W.O. Martins-Filho O.A. Cancado J.R. Pinto-Dias J.C. Brener Z. Gazzinelli G. Carvalho J.F. Colley D.G. Scand. J. Immunol. 1996; 43: 88-93Crossref PubMed Scopus (80) Google Scholar, 14Appay V. Dunbar P.R. Callan M. Klenerman P. Gillespie G.M. Papagno L. Ogg G.S. King A. Lechner F. Spina C.A. Little S. Havlir D.V. Richman D.D. Gruener N. Pape G. Waters A. Easterbrook P. Salio M. Cerundolo V. McMichael A.J. Rowland-Jones S.L. Nat. Med. 2002; 8: 379-385Crossref PubMed Scopus (1332) Google Scholar). CD4+CD28null T cells lack expression of CD154 and are therefore unable to provide help for B-cell proliferation and immunoglobulin production (21Weyand C.M. Brandes J.C. Schmidt D. Fulbright J.W. Goronzy J.J. Mech. Ageing Dev. 1998; 102: 131-147Crossref PubMed Scopus (158) Google Scholar). They have large cytoplasmic stores of interferon-γ (22Park W. Weyand C.M. Schmidt D. Goronzy J.J. Eur. J. Immunol. 1997; 27: 1082-1090Crossref PubMed Scopus (97) Google Scholar) and express CD161 (23Warrington K.J. Takemura S. Goronzy J.J. Weyand C.M. Arthritis Rheum. 2001; 44: 13-20Crossref PubMed Scopus (202) Google Scholar), giving them the potential to be inflammatory. They express a variety of receptors normally found on natural killer cells (24Snyder M.R. Muegge L.O. Offord C. O'Fallon W.M. Bajzer Z. Weyand C.M. Goronzy J.J. J. Immunol. 2002; 168: 3839-3846Crossref PubMed Scopus (95) Google Scholar). They have also acquired perforin and granzymes (34Javahery R. Khachi A. Lo K. Zenzie-Gregory B. Smale S.T. Mol. Cell. Biol. 1994; 14: 116-127Crossref PubMed Scopus (602) Google Scholar), which render CD4+CD28null T cells highly cytotoxic (25Nakajima T. Schulte S. Warrington K.J. Kopecky S.L. Frye R.L. Goronzy J.J. Weyand C.M. Circulation. 2002; 105: 570-575Crossref PubMed Scopus (306) Google Scholar). CD28null T cells may occupy ≥90% of the CD8 compartment of elderly persons (4Goronzy J.J. Fulbright J.W. Crowson C.S. Poland G.A. O'Fallon W.M. Weyand C.M. J. Virol. 2001; 75: 12182-12187Crossref PubMed Scopus (358) Google Scholar) and ≥40% of CD8+ T cells among patients with chronic inflammatory or infectious diseases (6Neil G.A. Summers R.W. Cheyne B.A. Carpenter C. Huang W.L. Waldschmidt T.J. Dig. Dis. Sci. 1994; 39: 1900-1908Crossref PubMed Scopus (17) Google Scholar, 9Honda M. Mengesha E. Albano S. Nichols W.S. Wallace D.J. Metzger A. Klinenberg J.R. Linker-Israeli M. Clin. Immunol. 2001; 99: 211-221Crossref PubMed Google Scholar, 10Schirmer M. Goldberger C. Wurzner R. Duftner C. Pfeiffer K.P. Clausen J. Neumayr G. Falkenbach A. Arthritis Res. 2002; 4: 71-76Crossref PubMed Scopus (96) Google Scholar, 11Dutra W.O. Martins-Filho O.A. Cancado J.R. Pinto-Dias J.C. Brener Z. Gazzinelli G. Carvalho J.F. Colley D.G. Scand. J. Immunol. 1996; 43: 88-93Crossref PubMed Scopus (80) Google Scholar, 12Schlienger K. Uyemura K. Jullien D. Sieling P.A. Rea T.H. Peter S. Modlin R.L. J. Immunol. 1998; 161: 2407-2413PubMed Google Scholar, 13Lewis D.E. Yang L. Luo W. Wang X. Rodgers J.R. AIDS. 1999; 13: 1029-1033Crossref PubMed Scopus (24) Google Scholar, 14Appay V. Dunbar P.R. Callan M. Klenerman P. Gillespie G.M. Papagno L. Ogg G.S. King A. Lechner F. Spina C.A. Little S. Havlir D.V. Richman D.D. Gruener N. Pape G. Waters A. Easterbrook P. Salio M. Cerundolo V. McMichael A.J. Rowland-Jones S.L. Nat. Med. 2002; 8: 379-385Crossref PubMed Scopus (1332) Google Scholar). In most cases, the loss of CD28 in the CD8 compartment is a terminal developmental stage because the majority of CD8+CD28null T cells have lost their proliferative capacity (18Spaulding C. Guo W. Effros R.B. Exp. Gerontol. 1999; 34: 633-644Crossref PubMed Scopus (210) Google Scholar, 26Lloyd T.E. Yang L. Tang D.N. Bennett T. Schober W. Lewis D.E. J. Immunol. 1997; 158: 1551-1558PubMed Google Scholar). Many of these cells also lack expression of CD11b; like their CD4+ counterparts, CD8+CD28null T cells have also acquired a variety of natural killer cell receptors, including CD56 and Fcγ receptor IIIA (27Zupo S. Azzoni L. Massara R. D'Amato A. Perussia B. Ferrarini M. J. Clin. Immunol. 1993; 13: 228-236Crossref PubMed Scopus (17) Google Scholar, 28Tarazona R. DelaRosa O. Alonso C. Ostos B. Espejo J. Pena J. Solana R. Mech. Ageing Dev. 2001; 121: 77-88Crossref Scopus (215) Google Scholar). Although some have retained their cytotoxic function, a large proportion of CD8+CD28null T cells are suppressors that specifically inhibit T-cell effectors as well antigen-presenting cells (29Cortesini R. Le Maoult J. Ciubotariu R. Cortesini N.S.F. Immunol. Rev. 2001; 182: 201-206Crossref PubMed Scopus (175) Google Scholar). Such gain and/or loss of function among the CD28null T cells are consistent with the idea that cellular senescence involves protection from apoptosis and the development of new phenotypes accompanying the limitation or cessation of proliferation (30Campisi J. Trends Cell Biol. 2001; 11: S27-S31Abstract Full Text PDF PubMed Scopus (721) Google Scholar). The novel phenotypes of these senescent lymphocytes may provide a basis for immunoincompetence during normal aging as well as increased severity of clinical manifestations of various chronic diseases that are correlated with the frequency of CD28null T cells (4Goronzy J.J. Fulbright J.W. Crowson C.S. Poland G.A. O'Fallon W.M. Weyand C.M. J. Virol. 2001; 75: 12182-12187Crossref PubMed Scopus (358) Google Scholar, 5Saurwein-Teissl M. Lung T.L. Marx F. Gschosser C. Asch E. Blasko I. Parson V. Bock V. Schonitzer V. Trannoy V. Grubeck-Loebenstein B. J. Immunol. 2002; 168: 5893-5899Crossref PubMed Scopus (422) Google Scholar, 7Martens P.B. Goronzy J.J. Schaid D.J. Weyand C.M. Arthritis Rheum. 1997; 40: 1106-1114Crossref PubMed Scopus (262) Google Scholar, 31Paul M.E. Shearer W.T. Kozinetz C.A. Lewis D.E. J. Clin. Allergy Immunol. 2001; 108: 258-264Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 32Khan N. Shariff N. Cobbold M. Bruton R. Ainsworth J.A. Sinclair A.J. Nayak L. Moss P.A.H. J. Immunol. 2002; 169: 1984-1992Crossref PubMed Scopus (617) Google Scholar). We reported previously that the loss of CD28 expression in T cells is associated with the inactivation of the transcriptional initiator (INR) of the gene promoter (3Vallejo A.N. Nestel A.R. Schirmer M. Weyand C.M. Goronzy J.J. J. Biol. Chem. 1998; 273: 8119-8129Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 33Vallejo A.N. Weyand C.M. Goronzy J.J. J. Biol. Chem. 2001; 276: 2565-2570Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The CD28 INR is a novel regulatory element that lacks homology with the consensus INR sequence (34Javahery R. Khachi A. Lo K. Zenzie-Gregory B. Smale S.T. Mol. Cell. Biol. 1994; 14: 116-127Crossref PubMed Scopus (602) Google Scholar). It consists of two contiguous sequence motifs that function as a unit (33Vallejo A.N. Weyand C.M. Goronzy J.J. J. Biol. Chem. 2001; 276: 2565-2570Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In vivo-derived CD28null T cells uniformly lack INR-specific transcription factors (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar), which explains why a CD28null phenotype is generally irreversible (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar, 24Snyder M.R. Muegge L.O. Offord C. O'Fallon W.M. Bajzer Z. Weyand C.M. Goronzy J.J. J. Immunol. 2002; 168: 3839-3846Crossref PubMed Scopus (95) Google Scholar, 35Namekawa T. Wagner U.G. Goronzy J.J. Weyand C.M. Arthritis Rheum. 1998; 41: 2108-2116Crossref PubMed Scopus (188) Google Scholar). Modulation in the levels of cell surface expression of CD28 during the proliferative life span of T cells is also coupled to INR activity (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar). Indeed, quantitative decreases in the levels of cell surface expression of CD28 and the ultimate generation of CD28null T cells in vitro by the continuous activation and passage of CD28+ precursors are accompanied by the repression of the INR (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar, 36Posnett D.N. Edinger J.W. Manavalan J.S. Irwin C. Marodon G. Int. Immunol. 1999; 11: 229-249Crossref PubMed Scopus (183) Google Scholar, 37Bryl E. Vallejo A.N. Weyand C.M. Goronzy J.J. J. Immunol. 2001; 167: 3231-3238Crossref PubMed Scopus (231) Google Scholar). To validate that CD28 INR function is a determinant of phenotypic transformations of T cells, we used a T-cell system in which the dynamics of CD28 expression could be rapidly assessed under controlled conditions. Because existing in vitro models involve highly protracted procedures (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar, 36Posnett D.N. Edinger J.W. Manavalan J.S. Irwin C. Marodon G. Int. Immunol. 1999; 11: 229-249Crossref PubMed Scopus (183) Google Scholar, 37Bryl E. Vallejo A.N. Weyand C.M. Goronzy J.J. J. Immunol. 2001; 167: 3231-3238Crossref PubMed Scopus (231) Google Scholar), a biological system that has an inducible but transient expression of CD28 is advantageous in dissecting molecular events controlling CD28+ to CD28null transition. Here, we show the usefulness of the T-cell lines JT and Jtag as models to track changes in CD28 expression. Studies were conducted to examine whether expression and/or loss of CD28 in a variety of situations are directly related to the functional competence of the CD28 INR. Cell Culture—JT cells were derived from Jtag (provided by Dr. David Spencer, Baylor College of Medicine, Houston, TX), a subline of Jurkat T cells that is stably transfected with SV40 large T-antigen (38Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2913-2917Abstract Full Text PDF Google Scholar). Jtag cells have very low levels of cell surface expression of CD28, but upregulate CD28 expression upon exposure to phorbol 12-myristate 13-acetate (PMA). This was in contrast to Jurkat cells, which expressed higher magnitudes of CD28 that were unaffected by PMA or other pharmacologic agents 2A. N. Vallejo, unpublished data. (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar, 20Vallejo A.N. Schirmer M. Weyand C.M. Goronzy J.J. J. Immunol. 2000; 165: 6301-6307Crossref PubMed Scopus (146) Google Scholar). To produce CD28null cells, Jtag cells were subjected to at least two rounds of flow cytometry selection for the complete lack of CD28 expression. The absence of CD28 was also confirmed by the lack of specific transcripts in standard reverse transcription (RT)-PCR assays for all the known splice variants of CD28 (19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar). The sorted CD28null cells were subsequently tested for the induction of CD28 expression with PMA by flow cytometry, resulting in the establishment of the CD28-inducible JT cell line. JT, Jtag, and Jurkat cells were cultured in RPMI 1640 medium (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) supplemented with 10% fetal calf serum (Summit Biotechnology, Fort Collins, CO), 2 mm l-glutamine, 50 units/ml penicillin, and 5 μg/ml streptomycin (Invitrogen). Cells were maintained at a density of 0.5–5.0 × 106 cells/ml in a humidified 5–7.5% CO2 tissue culture incubator. The murine P815 mastocytoma cell line (American Type Culture Collection, Manassas, VA) was also cultured in complete RPMI medium but incubated in 5% CO2. Wild-type P815 cell lines or those stably transfected with human CD80 or CD86 (provided by Dr. Lewis Lanier, University of California San Francisco; Ref. 39Azuma M. Cayabyab M. Buck D. Phillips J.H. Lanier L.L. J. Exp. Med. 1992; 175: 353-360Crossref PubMed Scopus (346) Google Scholar) were used as indicated in specific experiments. Flow Cytometry—CD28 and CD45 expression on JT, Jtag, and Jurkat cells were monitored by direct immunostaining with a fluorochrome-conjugated anti-human CD28 or anti-CD45 monoclonal antibody and analyzed by flow cytometry using a FACSCalibur cytometer (BD Biosciences) or a XL cytometer (Beckman Coulter). The kinetics of PMA-induced expression of CD28 on JT cells was examined. JT cells were incubated in PMA (Sigma Aldrich Corp.) at the indicated concentrations and CD28 expression was examined by flow cytometry after overnight incubation. The stability of CD28 expression after induction with PMA was also examined. PMA-stimulated JT cells were washed extensively and cultured in fresh medium, and CD28 expression was monitored during a 15- to 20-day culture period. Over this time, the cells remained viable with no significant levels of apoptosis as determined by propidium iodide staining and flow cytometry (data not shown). In other experiments, the influence of exogenous tumor necrosis factor (TNF)-α or CD28 ligation on the PMA-induced expression of CD28 on JT (or Jtag) cells was also examined. As indicated, 10 ng/ml recombinant human TNF-α (R&D Systems, Minneapolis, MN) was added to appropriate cultures during incubation with PMA or to PMA-stimulated cells cultured in fresh medium. In appropriate cultures of PMA-stimulated JT (or Jtag) cells, 2–5 μg/ml of anti-CD28 monoclonal antibody ANC28 (Calbiochem-EMD Biosciences), L293, or 28-2 (BD Biosciences Pharmingen), or an IgG isotype control was also added. In parallel experiments, PMA-treated JT (or Jtag) cells were cocultured with CD80+, CD86+, or control P815 cells at 5:1 JT/P815 cell ratio. After a 48 h-incubation, CD28 and CD45 expression were examined by flow cytometry. The levels of TNF-α receptor I and II were measured by indirect immunofluorescence staining and flow cytometry. Cells were stained with goat anti-TNF-α receptor I (R&D Systems) followed by a fluorochrome-conjugated rabbit anti-goat immunoglobulin (BD Biosciences), washed, then incubated with mouse monoclonal anti-TNF-α receptor II (R&D Systems) followed by rat anti-mouse IgG conjugated with a different fluorochrome (BD Biosciences). Reporter Gene Bioassays—The CD28 promoter-driven reporter plasmids p42 and p52 have been described previously (3Vallejo A.N. Nestel A.R. Schirmer M. Weyand C.M. Goronzy J.J. J. Biol. Chem. 1998; 273: 8119-8129Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Two additional luciferase reporters, KPN and RV, were constructed from a genomic clone of the 5′-flanking region of human CD28 gene (40Lee K.P. Taylor C. Petryniak B. Turka L.A. June C.H. Thompson C.B. J. Immunol. 1990; 145: 344-352PubMed Google Scholar; provided by Dr. Kelvin Lee, University of Miami Medical School, Miami, FL). The original clone was inserted in the pGEM-3Z vector (Promega, Madison, WI) at the EcoRI site and contained 1.7 kb extending into the first intron. DNA was digested with EcoRI and StuI, yielding an 812 bp-fragment that was subcloned into the pGL3 basic vector (Promega) at the SmaI site. The first 460 bp, corresponding to an Alu sequence, were deleted at the KpnI restriction site in the vector and in the CD28 promoter sequence, yielding the KPN construct. The RV construct was generated by restriction enzyme digestion at the KpnI site of the vector and the EcoRV site in the CD28 promoter sequence. DNA fragments were repaired using T4 DNA polymerase to generate blunt ends and were ligated to the pGL3 vector. Recombinant plasmids were used to transform DH5α E. coli hosts (Invitrogen Life Technologies). Plasmids were isolated using commercial kits, and DNA sequencing was performed to authenticate the sequence of the cloned CD28 promoter. The KPN reporter was a longer reporter construct than the RV, p42, and p52 reporters (Fig. 4B, diagram). It contained additional 5′ sequences flanking the minimal CD28 promoter sequences in the p42 and p52 reporters (3Vallejo A.N. Nestel A.R. Schirmer M. Weyand C.M. Goronzy J.J. J. Biol. Chem. 1998; 273: 8119-8129Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We had reported previously that the activity of the CD28 minimal promoter was regulated by the CD28 INR (33Vallejo A.N. Weyand C.M. Goronzy J.J. J. Biol. Chem. 2001; 276: 2565-2570Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), which functioned independently of a downstream GR element (41Lin C.J. Tam R.C. J. Immunol. 2001; 166: 6134-6143Crossref PubMed Scopus (16) Google Scholar). To further validate that the CD28 INR comprised the minimal promoter, the GR element was deleted in the RV construct, the shortest of the reporters. The KPN construct also lacked the GR element, whereas the p42 and p52 constructs have an intact GR element. Reporter plasmids were prepared using the EndoFree plasmid kit (Qiagen, Valencia, CA) and were used in transient transfection experiments with the GenePorter kit (Gene Therapy Systems, San Diego, CA). For each transfection, 4.5 × 106 JT cells were resuspended in 0.45 ml of serum-free RPMI 1640. A mixture of 8 μg of the appropriate luciferase reporter plasmid DNA, 1 μg of control pRL TK-Renilla reniformis luciferase (Promega), and 64 μl of the GenePorter reagent was added, and the final volume was adjusted to 2.5 ml with serum-free RPMI 1640. After 4 h of incubation, 2.5 ml of RPMI 1640 supplemented with 20% fetal calf serum was added and the transfectants were aliquoted to two tissue culture plates. PMA was added to one plate at a final concentration of 125 ng/ml. After 24 h, the cells were washed, resuspended in complete medium, and aliquoted into six 1-ml fractions in a 24-well culture plate. To the appropriate wells, 10 ng/ml TNF-α, 2 μg/ml anti-CD28 (ANC28, L293, or 28-2), or 2 μg/ml IgG isotype control was added. After an additional 48 h of incubation, CD28 expression was measured by flow cytometry, and luciferase activity was determined using the dual luciferase kit (Promega). Specific reporter luciferase activity was normalized against the internal R. reniformis luciferase activity and total protein concentration of cell lysates using a kit (BCA; Bio-Rad). Gel Shift and Transcription Assays—JT cells were exposed to PMA followed by the addition of 10 ng/ml TNF-α, 2–5 μg/ml anti-CD28 monoclonal antibody (ANC28, L293, or 28-2), 5 μg/ml IgG, or mouse P815 cells as indicated. Nuclear extracts were prepared and used in electrophoretic mobility shift assays (EMSA) using DNA-binding probes corresponding to the CD28 INR site α as described previously (3Vallejo A.N. Nestel A.R. Schirmer M. Weyand C.M. Goronzy J.J. J. Biol. Chem. 1998; 273: 8119-8129Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 19Vallejo A.N. Brandes J.C. Weyand C.M. Goronzy J.J. J. Immunol. 1999; 162: 6572-6579PubMed Google Scholar, 33Vallejo A.N. Weyand C.M. Goronzy J.J. J. Biol. Chem. 2001; 276: 2565-2570Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). As system controls, similar EMSAs were conducted using nuclear extracts from Jurkat T cells and the use of Sp1-binding oligonucleotides as probes. The nuclear extracts were also used in transcription assays to assess their competence in supporting transcription of CD28 INR-regulated templates. Transcription assays were conducted as described previously (33Vallejo A.N. Weyand C.M. Goronzy J.J. J. Biol. Chem. 2001; 276: 2565-2570Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 42Kaufmann J. Ahrens K. Koop R. Smale S.T. Muller R. Mol. Cell. Biol. 1998; 18: 233-239Crossref PubMed Scopus (54) Google Scholar). In brief, CD28 αβ-INR sequences were cloned at the 3′ flank of a canonical TATA box and upstream of a heterologous 180 bp G-less cassette. This template was incubated in a transcription reaction containing JT or Jurkat nuclear extracts and a ribonucleotide triphosphate mixture with [α-32P]UTP (Amersham Pharmacia Biotech). Transcription products were subjected to RNase T1 (Roche Molecular Biochemicals) digestion, extracted with phenol-chloroform, size fractionated on 8% polyacrylamide/6 m urea sequencing gels, and visualized by autoradiography. As system controls, similar transcription assays were conducted using Jurkat extracts as well as DNA templates that were under the control of the INR element of the terminal deoxyribonucleotidyl transferase (TdT) gene. RNase Protection Assay and RT-PCR—Jtag cells were incubated overnight with or without 125 ng/ml PMA. Cells were washed and resuspended in fresh medium. To appropriate cultures, TNF-α, anti-CD28 monoclonal antibody (ANC28 or 28-2), IgG control, or P815 cells were added as indicated. After 48 h, the cells were washed and total RNA was extracted using the RNAwiz reagent (Ambion, Austin, TX) and subjected to RNase protection assay (RPA) using the Ambion RPAIII kit. CD28 expression was detected by hybridization to a biotinylated R

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