Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients

BMC Immunology

Volumn 18, Issue 1, 2017, Pages

  Banas, Bernhard ^a   Böger, Carsten A ^a   Lückhoff, Gerhard ^b   Krüger, Bernd ^c   Barabas, Sascha ^d   Batzilla, Julia ^d   Schemmerer, Mathias ^d,e   Köstler, Josef ^e   Bendfeldt, Hanna ^d   Rascle, Anne ^d   Wagner, Ralf ^e   Deml, Ludwig ^d   Leicht, Joachim ^f   Krämer, Bernhard K ^c  

^a UNIVERSITY HOSPITAL REGENSBURG   (Germany)

^b Dialysis Center Landshut   (Germany)

^c UNIVERSITY OF HEIDELBERG   (Germany)

^d Lophius Biosciences   (Germany)

^e UNIVERSITY OF REGENSBURG   (Germany)

^f Dialysis Center Schwandorf   (Germany)

Author keywords

Cell mediated immunity; CMV; Cytomegalovirus; Hemodialysis; IE 1; IFN ELISpot; ITAg MHC Tetramers; Pp65; QuantiFERON CMV; T Track CMV

Indexed keywords

CYTOMEGALOVIRUS ANTIGEN PP65; T LYMPHOCYTE RECEPTOR; CYTOMEGALOVIRUS MATRIX PROTEIN 65KDA; IE1 PROTEIN, CYTOMEGALOVIRUS; IMMEDIATE EARLY PROTEIN; MATRIX PROTEIN; PHOSPHOPROTEIN; VIRUS ANTIBODY;

ADULT; AGED; ARTICLE; CELLULAR IMMUNITY; COHORT ANALYSIS; CONTROLLED CLINICAL TRIAL; CONTROLLED STUDY; CROSS REACTION; CROSS-SECTIONAL STUDY; CYTOMEGALOVIRUS; DIAGNOSTIC TEST ACCURACY STUDY; FEMALE; FUNCTIONAL ASSESSMENT; HEMODIALYSIS PATIENT; HUMAN; HUMAN CELL; HUMORAL IMMUNITY; IMMUNOASSAY; IMMUNOLOGY TEST KIT; KIDNEY TRANSPLANTATION; MAJOR CLINICAL STUDY; MAJOR HISTOCOMPATIBILITY COMPLEX; MALE; MULTICENTER STUDY; NONHUMAN; PATIENT MONITORING; PERFORMANCE; PROSPECTIVE STUDY; SEROLOGY; VALIDATION STUDY; BLOOD; CELL CULTURE; COMPARATIVE STUDY; CYTOMEGALOVIRUS INFECTIONS; HEMODIALYSIS; HOSPITAL ADMISSION; IMMUNOCOMPROMISED PATIENT; IMMUNOLOGICAL MONITORING; IMMUNOLOGY; KIDNEY FAILURE, CHRONIC; MIDDLE AGED; OBSERVER VARIATION; PROCEDURES; SENSITIVITY AND SPECIFICITY; VERY ELDERLY;

ADULT; AGED; AGED, 80 AND OVER; ANTIBODIES, VIRAL; CELLS, CULTURED; COHORT STUDIES; CYTOMEGALOVIRUS; CYTOMEGALOVIRUS INFECTIONS; FEMALE; HUMANS; IMMEDIATE-EARLY PROTEINS; IMMUNITY, CELLULAR; IMMUNOASSAY; IMMUNOCOMPROMISED HOST; KIDNEY FAILURE, CHRONIC; KIDNEY TRANSPLANTATION; MALE; MIDDLE AGED; MONITORING, IMMUNOLOGIC; OBSERVER VARIATION; PHOSPHOPROTEINS; RENAL DIALYSIS; SENSITIVITY AND SPECIFICITY; VIRAL MATRIX PROTEINS; WAITING LISTS;

EID: 85014597721     PISSN: None     EISSN: 14712172     Source Type: Journal    
DOI: 10.1186/s12865-017-0194-z     Document Type: Article

References (82)

1. Hanley PJ, Bollard CM. Controlling cytomegalovirus: helping the immune system take the lead. Viruses. 2014;6:2242-58.
2. Crough T, Khanna R. Immunobiology of human cytomegalovirus: from bench to bedside. Clin Microbiol Rev. 2009;22:76-98.
3. Schleiss MR. Cytomegalovirus in the neonate: immune correlates of infection and protection. Clin Dev Immunol. 2013;2013:501801.
4. Rossini G, Cerboni C, Santoni A, Landini MP, Landolfo S, Gatti D, et al. Interplay between human cytomegalovirus and intrinsic/innate host responses: a complex bidirectional relationship. Mediators Inflamm. 2012;2012:607276.
5. Jeitziner SM, Walton SM, Torti N, Oxenius A. Adoptive transfer of cytomegalovirus-specific effector CD4+ T cells provides antiviral protection from murine CMV infection. Eur J Immunol. 2013;43:2886-95.
6. Sester M, Sester U, Gärtner B, Heine G, Girndt M, Mueller-Lantzsch N, et al. Levels of virus-specific CD4 T cells correlate with cytomegalovirus control and predict virus-induced disease after renal transplantation. Transplantation. 2001;71:1287-94.
7. Gamadia LE, Remmerswaal EBM, Weel JF, Bemelman F, van Lier RAW, Ten Berge IJM. Primary immune responses to human CMV: a critical role for IFN-gamma-producing CD4+ T cells in protection against CMV disease. Blood. 2003;101:2686-92.
8. Sester M, Sester U, Gärtner BC, Girndt M, Meyerhans A, Köhler H. Dominance of virus-specific CD8 T cells in human primary cytomegalovirus infection. J Am Soc Nephrol JASN. 2002;13:2577-84.
9. Widmann T, Sester U, Gärtner BC, Schubert J, Pfreundschuh M, Köhler H, et al. Levels of CMV specific CD4 T cells are dynamic and correlate with CMV viremia after allogeneic stem cell transplantation. PLoS One. 2008;3:e3634.
10. Sacre K, Carcelain G, Cassoux N, Fillet A-M, Costagliola D, Vittecoq D, et al. Repertoire, diversity, and differentiation of specific CD8 T cells are associated with immune protection against human cytomegalovirus disease. J Exp Med. 2005;201:1999-2010.
11. Sester M, Sester U, Gärtner B, Kubuschok B, Girndt M, Meyerhans A, et al. Sustained high frequencies of specific CD4 T cells restricted to a single persistent virus. J Virol. 2002;76:3748-55.
12. Dunn HS, Haney DJ, Ghanekar SA, Stepick-Biek P, Lewis DB, Maecker HT. Dynamics of CD4 and CD8 T Cell Responses to Cytomegalovirus in Healthy Human Donors. J Infect Dis. 2002;186:15-22.
13. Sinclair E, Black D, Epling CL, Carvidi A, Josefowicz SZ, Bredt BM, et al. CMV antigen-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation responses in healthy CMV-seropositive individuals. Viral Immunol. 2004;17:445-54.
14. Sester U, Presser D, Dirks J, Gärtner BC, Köhler H, Sester M. PD-1 expression and IL-2 loss of cytomegalovirus- specific T cells correlates with viremia and reversible functional anergy. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2008;8:1486-97.
15. Dirks J, Tas H, Schmidt T, Kirsch S, Gärtner BC, Sester U, et al. PD-1 analysis on CD28(-) CD27(-) CD4 T cells allows stimulation-independent assessment of CMV viremic episodes in transplant recipients. Off J Am Soc Transplant Am Soc Transpl Surg. 2013;13:3132-41.
16. Engstrand M, Lidehall AK, Totterman TH, Herrman B, Eriksson B-M, Korsgren O. Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment. Clin Exp Immunol. 2003;132:96-104.
17. Huster KM, Stemberger C, Gasteiger G, Kastenmüller W, Drexler I, Busch DH. Cutting edge: memory CD8 T cell compartment grows in size with immunological experience but nevertheless can lose function. J Immunol. 2009;183:6898-902.
18. Sund F, Lidehäll A-K, Claesson K, Foss A, Tötterman TH, Korsgren O, et al. CMV-specific T-cell immunity, viral load, and clinical outcome in seropositive renal transplant recipients: a pilot study. Clin Transplant. 2010;24:401-9.
19. Fernández-Ruiz M, Kumar D, Humar A. Clinical immune-monitoring strategies for predicting infection risk in solid organ transplantation. Clin Transl Immunol. 2014;3:e12.
20. Kotton CN. Management of cytomegalovirus infection in solid organ transplantation. Nat Rev Nephrol. 2010;6:711-21.
21. Lisboa LF, Kumar D, Wilson LE, Humar A. Clinical utility of cytomegalovirus cell-mediated immunity in transplant recipients with cytomegalovirus viremia. Transplantation. 2012;93:195-200.
22. Koehl U, Dirkwinkel E, Koenig M, Erben S, Soerensen J, Bader P, et al. Reconstitution of cytomegalovirus specific T cells after pediatric allogeneic stem cell transplantation: results from a pilot study using a multi-allele CMV tetramer group. Klin Padiatr. 2008;220:348-52.
23. Gratama JW, Boeckh M, Nakamura R, Cornelissen JJ, Brooimans RA, Zaia JA, et al. Immune monitoring with iTAg MHC Tetramers for prediction of recurrent or persistent cytomegalovirus infection or disease in allogeneic hematopoietic stem cell transplant recipients: a prospective multicenter study. Blood. 2010;116:1655-62.
24. Klenerman P, Cerundolo V, Dunbar PR. Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol. 2002;2:263-72.
25. Bunde T, Kirchner A, Hoffmeister B, Habedank D, Hetzer R, Cherepnev G, et al. Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells. J Exp Med. 2005;201:1031-6.
26. Egli A, Binet I, Binggeli S, Jäger C, Dumoulin A, Schaub S, et al. Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients. J Transl Med. 2008;6:29.
27. Abate D, Saldan A, Mengoli C, Fiscon M, Silvestre C, Fallico L, et al. Comparison of cytomegalovirus (CMV) enzyme-linked immunosorbent spot and CMV quantiferon gamma interferon-releasing assays in assessing risk of CMV infection in kidney transplant recipients. J Clin Microbiol. 2013;51:2501-7.
28. Manuel O, Husain S, Kumar D, Zayas C, Mawhorter S, Levi ME, et al. Assessment of cytomegalovirus-specific cell-mediated immunity for the prediction of cytomegalovirus disease in high-risk solid-organ transplant recipients: a multicenter cohort study. Clin Infect Dis Off Publ Infect Dis Soc Am. 2013;56:817-24.
29. Cantisán S, Lara R, Montejo M, Redel J, Rodríguez-Benot A, Gutiérrez-Aroca J, et al. Pretransplant interferon-γ secretion by CMV-specific CD8+ T cells informs the risk of CMV replication after transplantation. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2013;13:738-45.
30. Kumar D, Chernenko S, Moussa G, Cobos I, Manuel O, Preiksaitis J, et al. Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2009;9:1214-22.
31. Bestard O, Lucia M, Crespo E, Van Liempt B, Palacio D, Melilli E, et al. Pretransplant immediately early-1-specific T cell responses provide protection for CMV infection after kidney transplantation. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2013;13:1793-805.
32. Godard B, Gazagne A, Gey A, Baptiste M, Vingert B, Pegaz-Fiornet B, et al. Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes. Hum Immunol. 2004;65:1307-18.
33. Schmittel A, Keilholz U, Scheibenbogen C. Evaluation of the interferon-gamma ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood. J Immunol Methods. 1997;210:167-74.
34. Walker S, Fazou C, Crough T, Holdsworth R, Kiely P, Veale M, et al. Ex vivo monitoring of human cytomegalovirus-specific CD8+ T-cell responses using QuantiFERON-CMV. Transpl Infect Dis Off J Transplant Soc. 2007;9:165-70.
35. Clari MÁ, Muñoz-Cobo B, Solano C, Benet I, Costa E, Remigia MJ, et al. Performance of the QuantiFERON-cytomegalovirus (CMV) assay for detection and estimation of the magnitude and functionality of the CMV-specific gamma interferon-producing CD8(+) T-cell response in allogeneic stem cell transplant recipients. Clin Vaccine Immunol CVI. 2012;19:791-6.
36. Fleming T, Dunne J, Crowley B. Ex vivo monitoring of human cytomegalovirus-specific CD8(+) T-Cell responses using the QuantiFERON-CMV assay in allogeneic hematopoietic stem cell transplant recipients attending an Irish hospital. J Med Virol. 2010;82:433-40.
37. Barabas S, Gary R, Bauer T, Lindner J, Lindner P, Weinberger B, et al. Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein. PLoS Pathog. 2008;4:e1000198.
38. Barabas S, Spindler T, Kiener R, Tonar C, Lugner T, Batzilla J, Bendfeldt H, Rascle A, Asbach B, Wagner R, Deml L. An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity. BMC Immunol. 2017. doi: 10.1186/s12865-017-0195-y.
39. Reuschel E, Barabas S, Zeman F, Bendfeldt H, Rascle A, Deml L, et al. Functional impairment of CMV-reactive cellular immunity during pregnancy. J Med Virol. 2017;89:324-31.
40. Bui H-H, Sidney J, Dinh K, Southwood S, Newman MJ, Sette A. Predicting population coverage of T-cell epitope-based diagnostics and vaccines. BMC Bioinformatics. 2006;7:153.
41. van Dommelen SLH, Tabarias HA, Smyth MJ, Degli-Esposti MA. Activation of Natural Killer (NK) T Cells during Murine Cytomegalovirus Infection Enhances the Antiviral Response Mediated by NK Cells. J Virol. 2003;77:1877-84.
42. Sylwester AW, Mitchell BL, Edgar JB, Taormina C, Pelte C, Ruchti F, et al. Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med. 2005;202:673-85.
43. Kamath AT, Sheasby CE, Tough DF. Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma. J Immunol. 2005;174:767-76.
44. Min-Oo G, Lanier LL. Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection. J Exp Med. 2014;211:2669-80.
45. Reschner A, Hubert P, Delvenne P, Boniver J, Jacobs N. Innate lymphocyte and dendritic cell cross-talk: a key factor in the regulation of the immune response. Clin Exp Immunol. 2008;152:219-26.
46. Ferlazzo G, Morandi B. Cross-Talks between Natural Killer Cells and Distinct Subsets of Dendritic Cells. Front Immunol. 2014;5:159.
47. Kern F, Surel IP, Faulhaber N, Frömmel C, Schneider-Mergener J, Schönemann C, et al. Target structures of the CD8(+)-T-cell response to human cytomegalovirus: the 72-kilodalton major immediate-early protein revisited. J Virol. 1999;73:8179-84.
48. Litjens NHR, Huisman M, van den Dorpel M, Betjes MGH. Impaired immune responses and antigen-specific memory CD4+ T cells in hemodialysis patients. J Am Soc Nephrol JASN. 2008;19:1483-90.
49. Girndt M, Sester U, Sester M, Kaul H, Köhler H. Impaired cellular immune function in patients with end-stage renal failure. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc. 1999;14:2807-10.
50. Girndt M, Sester M, Sester U, Kaul H, Köhler H. Defective expression of B7-2 (CD86) on monocytes of dialysis patients correlates to the uremia-associated immune defect. Kidney Int. 2001;59:1382-9.
51. Lonnemann G. Impaired NK, cell function in ESRD patients. Blood Purif. 2008;26:315-6.
52. Mattes FM, Vargas A, Kopycinski J, Hainsworth EG, Sweny P, Nebbia G, et al. Functional impairment of cytomegalovirus specific CD8 T cells predicts high-level replication after renal transplantation. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2008;8:990-9.
53. Gerna G, Lilleri D, Fornara C, Comolli G, Lozza L, Campana C, et al. Monitoring of human cytomegalovirus-specific CD4 and CD8 T-cell immunity in patients receiving solid organ transplantation. Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2006;6:2356-64.
54. Tabi Z, Moutaftsi M, Borysiewicz LK. Human cytomegalovirus pp 65- and immediate early 1 antigen-specific HLA class I-restricted cytotoxic T cell responses induced by cross-presentation of viral antigens. J Immunol. 2001;166:5695-703.
55. Khan N, Bruton R, Taylor GS, Cobbold M, Jones TR, Rickinson AB, et al. Identification of cytomegalovirus-specific cytotoxic T lymphocytes in vitro is greatly enhanced by the use of recombinant virus lacking the US2 to US11 region or modified vaccinia virus Ankara expressing individual viral genes. J Virol. 2005;79:2869-79.
56. Manley TJ, Luy L, Jones T, Boeckh M, Mutimer H, Riddell SR. Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection. Blood. 2004;104:1075-82.
57. Gilbert MJ, Riddell SR, Li CR, Greenberg PD. Selective interference with class I major histocompatibility complex presentation of the major immediate-early protein following infection with human cytomegalovirus. J Virol. 1993;67:3461-9.
58. Scheller N, Furtwängler R, Sester U, Maier R, Breinig T, Meyerhans A. Human cytomegalovirus protein pp 65: an efficient protein carrier system into human dendritic cells. Gene Ther. 2008;15:318-25.
59. Delmas S, Martin L, Baron M, Nelson JA, Streblow DN, Davignon J-L. Optimization of CD4+ T lymphocyte response to human cytomegalovirus nuclear IE1 protein through modifications of both size and cellular localization. J Immunol. 2005;175:6812-9.
60. Khan N, Cobbold M, Keenan R, Moss PAH. Comparative analysis of CD8+ T cell responses against human cytomegalovirus proteins pp65 and immediate early 1 shows similarities in precursor frequency, oligoclonality, and phenotype. J Infect Dis. 2002;185:1025-34.
61. Forner G, Saldan A, Mengoli C, Gussetti N, Palù G, Abate D. CMV-ELISPOT but not CMV-QuantiFERON assay is a novel biomarker to determine risk of congenital CMV infection in pregnant women. J Clin Microbiol. 2016;54(8):2149-54.
62. Saldan A, Forner G, Mengoli C, Tinto D, Fallico L, Peracchi M, et al. Comparison of cell-mediated immune assays CMV-ELISPOT and CMV-QuantiFERON in CMV seropositive and seronegative pregnant and non-pregnant women. J Clin Microbiol. 2016;54(5):1352-6.
63. Loeth N, Assing K, Madsen HO, Vindeløv L, Buus S, Stryhn A. Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors. PLoS One. 2012;7:e31420.
64. Lúcia M, Crespo E, Melilli E, Cruzado JM, Luque S, Llaudó I, et al. Preformed frequencies of cytomegalovirus (CMV)-specific memory T and B cells identify protected CMV-sensitized individuals among seronegative kidney transplant recipients. Clin Infect Dis Off Publ Infect Dis Soc Am. 2014;59:1537-45.
65. Sester M, Gärtner BC, Sester U, Girndt M, Mueller-Lantzsch N, Köhler H. Is the cytomegalovirus serologic status always accurate? A comparative analysis of humoral and cellular immunity. Transplantation. 2003;76:1229-30.
66. Edson CM, Hosler BA, Respess RA, Waters DJ, Thorley-Lawson DA. Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein. J Virol. 1985;56:333-6.
67. Vafai A, Wroblewska Z, Graf L. Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein. Virus Res. 1990;15:163-74.
68. Su LF, Kidd BA, Han A, Kotzin JJ, Davis MM. Virus-specific CD4(+) memory-phenotype T cells are abundant in unexposed adults. Immunity. 2013;38:373-83.
69. Kieper WC, Troy A, Burghardt JT, Ramsey C, Lee JY, Jiang H-Q, et al. Recent immune status determines the source of antigens that drive homeostatic T cell expansion. J Immunol. 2005;174:3158-63.
70. Yukl SA, Shergill AK, Girling V, Li Q, Killian M, Epling L, et al. Site-specific differences in T cell frequencies and phenotypes in the blood and gut of HIV-uninfected and ART-treated HIV+ adults. PLoS One. 2015;10:e0121290.
71. Dubois E, Ruschil C, Bischof F. Low frequencies of central memory CD4 T cells in progressive multifocal leukoencephalopathy. Neurol Neuroimmunol Neuroinflammation [Internet]. 2015. [cited Oct 24 2016];2. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630684/.
72. Alanio C, Lemaitre F, Law HKW, Hasan M, Albert ML. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Blood. 2010;115:3718-25.
73. Sprent J, Surh CD. Normal T cell homeostasis: the conversion of naive cells into memory-phenotype cells. Nat Immunol. 2011;12:478-84.
74. Hanley PJ, Melenhorst JJ, Nikiforow S, Scheinberg P, Blaney JW, Demmler-Harrison G, et al. CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo. Sci Transl Med. 2015;7:285ra63.
75. Hanley PJ, Shaffer DR, Cruz CRY, Ku S, Tzou B, Liu H, et al. Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation. Cytotherapy. 2011;13:976-86.
76. Brenchley JM, Douek DC, Ambrozak DR, Chatterji M, Betts MR, Davis LS, et al. Expansion of activated human naïve T-cells precedes effector function. Clin Exp Immunol. 2002;130:432-40.
77. Maroof A, Beattie L, Kirby A, Coles M, Kaye PM. Dendritic cells matured by inflammation induce CD86-dependent priming of naive CD8+ T cells in the absence of their cognate peptide antigen. J Immunol. 2009;183:7095-103.
78. Zippelius A, Pittet MJ, Batard P, Rufer N, de Smedt M, Guillaume P, et al. Thymic selection generates a large T cell pool recognizing a self-peptide in humans. J Exp Med. 2002;195:485-94.
79. Whitmire JK, Eam B, Whitton JL. Tentative T cells: memory cells are quick to respond, but slow to divide. PLoS Pathog. 2008;4:e1000041.
80. Veiga-Fernandes H, Walter U, Bourgeois C, McLean A, Rocha B. Response of naïve and memory CD8+ T cells to antigen stimulation in vivo. Nat Immunol. 2000;1:47-53.
81. Brehm MA, Mangada J, Markees TG, Pearson T, Daniels KA, Thornley TB, et al. Rapid quantification of naive alloreactive T cells by TNF-alpha production and correlation with allograft rejection in mice. Blood. 2007;109:819-26.
82. Ben-Sasson SZ, Gerstel R, Hu-Li J, Paul WE. Cell division is not a "clock" measuring acquisition of competence to produce IFN-gamma or IL-4. J Immunol. 2001;166:112-20.