Cytokine flow cytometry: a multiparametric approach for assessing cellular immune responses to viral antigens.

Considerable attention has been focused on CD8 and CD4 T cell responses as a major element of the cellular immune response to viral infections including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, increasing evidence based on the recent introduction of more quantitative assays for measuring antigen-specific T cells has suggested that the role of these cells in the development of a protective immune response to a particular viral pathogen may be determined by a complex interplay of multiple virologic and cellular factors. Thus, measurements of only the frequencies of the T cell subsets participating in the response to viral pathogens may be an incomplete reflection of efficacy. In this review, we suggest that some measurable factors may influence the role of T cell immunity in conferring protection, including functional avidity, epitope breadth and specificity, proliferative capacity, cytokine repertoire, degree of anergy, and differentiation phenotype, as well as magnitude, of viral-specific CD4 and CD8 T cells. We suggest that automated cytokine flow cytometry (CFC) is an efficient approach to the measurement of the complex interplay of multiple immune parameters involved in immune protection. These ideas are discussed in the context of new developments in sample preparation and analysis automation.

[1]  J. Altman,et al.  Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. , 1998, Immunity.

[2]  Philip J. R. Goulder,et al.  Phenotypic Analysis of Antigen-Specific T Lymphocytes , 1996, Science.

[3]  J. Heeney.  The critical role of CD4(+) T-cell help in immunity to HIV. , 2002, Vaccine.

[4]  V. Jansen,et al.  Effector cytotoxic T lymphocyte numbers induced by vaccination should exceed levels in chronic infection for protection from HIV. , 2001, Vaccine.

[5]  E. Schiff,et al.  Hepatitis C: Diagnostic Assays , 1995, Seminars in liver disease.

[6]  E. Rosenberg,et al.  Immune control of HIV-1 after early treatment of acute infection , 2000, Nature.

[7]  C. Pitcher,et al.  Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. , 1997, The Journal of clinical investigation.

[8]  J. Mellors,et al.  CD8+ T-Cell Gamma Interferon Production Specific for Human Immunodeficiency Virus Type 1 (HIV-1) in HIV-1-Infected Subjects , 2000, Clinical Diagnostic Laboratory Immunology.

[9]  Noel R. Rose,et al.  Manual of clinical laboratory immunology , 2002 .

[10]  L. Weinberger,et al.  Dramatic Rise in Plasma Viremia after CD8+ T Cell Depletion in Simian Immunodeficiency Virus–infected Macaques , 1999, The Journal of experimental medicine.

[11]  V. Maino,et al.  Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. , 1998, Journal of immunological methods.

[12]  F. Valentine,et al.  Lymphocytes from some long-term seronegative heterosexual partners of HIV-infected individuals proliferate in response to HIV antigens. , 1992, AIDS research and human retroviruses.

[13]  J. Strominger,et al.  Immune correlates of protection from HIV and AIDS - more answers but yet more questions. , 1999, Immunology today.

[14]  D. Ho,et al.  Normal T-Cell Turnover in Sooty Mangabeys Harboring Active Simian Immunodeficiency Virus Infection , 2000, Journal of Virology.

[15]  J. Lisziewicz,et al.  Control of SIV rebound through structured treatment interruptions during early infection. , 2000, Science.

[16]  R. Johnson,et al.  Mechanisms of protection against simian immunodeficiency virus infection. , 2002, Vaccine.

[17]  V. Maino,et al.  Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. , 1998, Cytometry.

[18]  Hengel Rl,et al.  An update on the use of flow cytometry in HIV infection and AIDS. , 2001 .

[19]  Holden T Maecker,et al.  Dynamics of CD4 and CD8 T cell responses to cytomegalovirus in healthy human donors. , 2002, The Journal of infectious diseases.

[20]  A. Osterhaus,et al.  The advantage of early recognition of HIV-infected cells by cytotoxic T-lymphocytes. , 2002, Vaccine.

[21]  V. Maino,et al.  Simultaneous detection of DNA synthesis and cytokine production in staphylococcal enterotoxin B activated CD4+ T lymphocytes by flow cytometry. , 1997, Journal of immunological methods.

[22]  D. Strick,et al.  Comprehensive Epitope Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Responses Directed against the Entire Expressed HIV-1 Genome Demonstrate Broadly Directed Responses, but No Correlation to Viral Load , 2003, Journal of Virology.

[23]  B. Gärtner,et al.  The fraction of perforin-expressing HIV-specific CD8 T cells is a marker for disease progression in HIV infection , 2002, AIDS.

[24]  C. Pitcher,et al.  Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. , 2001, Journal of immunological methods.

[25]  O Ouchterlony,et al.  A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. , 1983, Journal of immunological methods.

[26]  K. Fujii,et al.  Rapid Determination of Epstein-Barr Virus–Specific CD8+T-Cell Frequencies by Flow Cytometry , 1999 .

[27]  Holden T Maecker,et al.  Performance of plate-based cytokine flow cytometry with automated data analysis , 2003, BMC Immunology.

[28]  J. McCune,et al.  Loss of cytomegalovirus-specific CD4+ T cell responses in human immunodeficiency virus type 1-infected patients with high CD4+ T cell counts and recurrent retinitis. , 2001, The Journal of infectious diseases.

[29]  R. Gehrz,et al.  Characterization of the role of mononuclear cell subpopulations in the in vitro lymphocyte proliferation assay. , 1979, Clinical and experimental immunology.

[30]  Fred Wang,et al.  Direct Relationship between Suppression of Virus-Specific Immunity and Emergence of Cytomegalovirus Disease in Simian AIDS , 2003, Journal of Virology.

[31]  R. Davey,et al.  Laboratory methods in the diagnosis and prognostic staging of infection with human immunodeficiency virus type 1. , 1990, Reviews of infectious diseases.

[32]  R. Ahmed,et al.  Long-Term CD4 Th1 and Th2 Memory following Acute Lymphocytic Choriomeningitis Virus Infection , 1998, Journal of Virology.

[33]  V. Maino,et al.  The CD4(+) T cell response to HIV-1. , 2000, Current opinion in immunology.

[34]  Alan S. Perelson,et al.  Quantification of Cell Turnover Kinetics Using 5-Bromo-2′-deoxyuridine1 , 2000, The Journal of Immunology.

[35]  Alan S. Perelson,et al.  Turnover of CD4+ and CD8+ T Lymphocytes in HIV-1 Infection as Measured by Ki-67 Antigen , 1998, The Journal of experimental medicine.

[36]  G. Rutherford,et al.  Surveillance for Incident HIV Infection: New Technology and New Opportunities , 2000, Journal of acquired immune deficiency syndromes.

[37]  E. Rosenberg,et al.  Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. , 1997, Science.

[38]  V. Maino,et al.  Enhancement of HIV type 1 antigen-specific CD4+ T cell memory in subjects with chronic HIV type 1 infection receiving an HIV type 1 immunogen. , 2000, AIDS research and human retroviruses.

[39]  Circulating CD8 T Lymphocytes in Human Immunodeficiency Virus-Infected Individuals Have Impaired Function and Downmodulate CD3ζ, the Signaling Chain of the T-Cell Receptor Complex , 1998 .

[40]  P. Limburg,et al.  In vitro cytokine production and proliferation of T cells from patients with anti-proteinase 3- and antimyeloperoxidase-associated vasculitis, in response to proteinase 3 and myeloperoxidase. , 2002, Arthritis and rheumatism.

[41]  James G. Herndon,et al.  Control of a Mucosal Challenge and Prevention of AIDS by a Multiprotein DNA/MVA Vaccine , 2001, Science.

[42]  H. Farzadegan.  HIV-1 antibodies and serology. , 1994, Clinics in laboratory medicine.

[43]  S. Rowland-Jones,et al.  Direct visualization of HIV-1-specific cytotoxic T lymphocytes during primary infection , 2000, AIDS.

[44]  G. Dworacki,et al.  Correlation between DNA Content, Expression of Ki-67 Antigen of Tumor Cells and Immunophenotype of Lymphocytes from Malignant Pleural Effusions , 1998, Tumor Biology.

[45]  Ghanekar Sa,et al.  Cytokine flow cytometry: multiparametric approach to immune function analysis. , 2003 .

[46]  P. Schmid‐Grendelmeier,et al.  Altered intracellular expression of the chemokines MIP‐1α, MIP‐1β and IL‐8 by peripheral blood CD4+ and CD8+ T cells in mild allergic asthma , 2003, Allergy.

[47]  Decreased HIV-specific CD4 T cell proliferation in long-term HIV-infected individuals on antiretroviral therapy. , 2001, AIDS.

[48]  V. Maino,et al.  CD4+CD8dim T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV‐1 antigens , 2001, European journal of immunology.

[49]  Z. Toossi,et al.  Dysregulation of β-chemokines in the lungs of HIV-1-infected patients , 2001 .

[50]  D. Nixon,et al.  Rapid quantification of SIV-specific CD8 T cell responses with recombinant vaccinia virus ELISPOT or cytokine flow cytometry. , 2000, AIDS.

[51]  I. Elovaara,et al.  Differential intracellular expression of CCR5 and chemokines in multiple sclerosis subtypes , 2002, Journal of Neurology.

[52]  M. Atkins,et al.  Immunologic Monitoring of Cancer Vaccine Therapy: Results of a Workshop Sponsored by the Society for Biological Therapy , 2002, Journal of immunotherapy.

[53]  N. Sueyoshi,et al.  Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity. , 2002, Biochemical and biophysical research communications.

[54]  J. McCune,et al.  Restoration of cytomegalovirus-specific CD4+ T-lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with HIV-1 , 1998, Nature Medicine.

[55]  S. Gurunathan,et al.  Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection , 1998, Nature Medicine.

[56]  E. Rosenberg,et al.  Identification of Dominant Optimal HLA-B60- and HLA-B61-Restricted Cytotoxic T-Lymphocyte (CTL) Epitopes: Rapid Characterization of CTL Responses by Enzyme-Linked Immunospot Assay , 2000, Journal of Virology.

[57]  B. Walker,et al.  Persistent HIV-1-specific CTL clonal expansion despite high viral burden post in utero HIV-1 infection. , 1999, Journal of immunology.

[58]  J. Clot,et al.  Perforin expression in T cells and virological response to PEG-interferon alpha2b in HIV-1 infection , 2003, AIDS.

[59]  E. Rosenberg,et al.  Rapid Definition of Five Novel HLA-A∗3002-Restricted Human Immunodeficiency Virus-Specific Cytotoxic T-Lymphocyte Epitopes by Elispot and Intracellular Cytokine Staining Assays , 2001, Journal of Virology.

[60]  A. Saito,et al.  Lymphocyte-proliferative responses to HIV antigens as a potential measure of immunological reconstitution in HIV disease. , 1998, AIDS research and human retroviruses.

[61]  D. Fulcher,et al.  Carboxyfluorescein succinimidyl ester‐based proliferative assays for assessment of T cell function in the diagnostic laboratory , 1999, Immunology and cell biology.

[62]  V. Maino,et al.  T cell immunity to HIV: defining parameters of protection. , 2003, Current HIV Research.

[63]  V. Maino,et al.  Gamma Interferon Expression in CD8+ T Cells Is a Marker for Circulating Cytotoxic T Lymphocytes That Recognize an HLA A2-Restricted Epitope of Human Cytomegalovirus Phosphoprotein pp65 , 2001, Clinical Diagnostic Laboratory Immunology.

[64]  H. Maecker,et al.  Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells. , 2000, Cytometry.

[65]  V. Maino,et al.  Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood. , 2001, Viral immunology.

[66]  K. Ohshima,et al.  Perforin and Fas pathways of cytotoxic T‐cells in histiocytic necrotizing lymphadenitis , 1998, Histopathology.

[67]  M. Altfeld,et al.  Less is more? STI in acute and chronic HIV-1 infection , 2001, Nature Medicine.

[68]  M. Baseler,et al.  Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity. , 2001, Immunology letters.

[69]  C. Gray,et al.  The ELISPOT Assay: An Easily Transferable Method for Measuring Cellular Responses and Identifying T Cell Epitopes , 2002, Clinical chemistry and laboratory medicine.

[70]  M. Jacobson,et al.  Immunological and virological surrogate markers in the evaluation of therapies for HIV infection. , 1990, AIDS (London).

[71]  W R Clark,et al.  Role of TNF-alpha in CD8+ cytotoxic T lymphocyte-mediated lysis. , 1993, Journal of immunology.

[72]  D. Nixon,et al.  Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects. , 2001, Virology.

[73]  S. Hoffman,et al.  ELISPOT assay for detection of peptide specific Interferon-γ secreting cells in rhesus macaques , 2001 .