Skip to main content
SpringerLink
Log in
Book cover

Immunotoxicity Testing pp 199–208Cite as

Evaluation of Cell-Mediated Immune Function Using the Cytotoxic T-Lymphocyte Assay

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1803))

Abstract

Evaluation of cell-mediated immunity (CMI) is a significant component in any assessment designed to predict the full range of potential immunotoxic risk underlying health risks. Among measures of CMI, the cytotoxic T-lymphocyte (CTL) response is recognized as perhaps the most relevant functional measure that reflects cell-mediated acquired immune defense against viral infections and cancer. The CTL response against T-dependent antigens requires the cooperation of at least three different major categories of immune cells. These include professional antigen-presenting cells (e.g., dendritic cells), CD4+ T helper lymphocytes, and CD8+ T effector lymphocytes. It is also among the few functional responses dependent on and, hence, capable of evaluating effective antigen presentation via both class I and class II molecules of the major histocompatibility complex (MHC). For this reason, the CTL assay is an excellent candidate for evaluation of potential immunotoxicity. This chapter provides an example of a mouse CTL assay against influenza virus that has been utilized for this purpose.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bretscher PA (1981) Significance and mechanisms of the cellular regulation of the immune response. Fed Proc 40:1473–1478

    PubMed  CAS  Google Scholar 

  2. Asherson CR, Stone SH (1962) Selective and specific inhibition of 24 hour skin reactions in the guinea pig. I. Immune deviation: description of the phenomenon and the effect of splenectomy. Immunology 9:205–217

    Google Scholar 

  3. Parish CR (1972) The relationship between humoral and cell-mediated immunity. Transplant Rev 13:35–66

    PubMed  CAS  Google Scholar 

  4. Ramshaw IA, Bretscher PA, Parish CR (1976) Regulation of the immune response. I. Suppression of delayed-type hypersensitivity by T cells from mice expressing humoral immunity. Eur J Immunol 6:674–679

    Article  PubMed  CAS  Google Scholar 

  5. Ramshaw IA, Bretscher PA, McKenzie IFC (1977) Discrimination of suppressor T cells of humoral and cell-mediated immunity by anti-Ly and anti-Ia sera. Cell Immunol 31:364–369

    Article  PubMed  CAS  Google Scholar 

  6. Bretscher PA (1983) In vitro analysis of the cellular interactions between unprimed lymphocytes responsible for determining the class of response an antigen induces: specific T cells switch a cell-mediated response to a humoral response. J Immunol 131:1103–1107

    PubMed  CAS  Google Scholar 

  7. Bretscher PA (1983) Regulation of the immune response induced by antigen. I. Specific T cells switch the in vivo immune response from a cell-mediated to a humoral mode. Cell Immunol 81:345–356

    Article  PubMed  CAS  Google Scholar 

  8. Tuttosi S, Bretscher PA (1992) Antigen-specific CD8+ T cells switch the immune response induced by antigen from an IgG to a cell-mediated mode. J Immunol 148:397–403

    PubMed  CAS  Google Scholar 

  9. Ramshaw IA, Bretscher PA, Parish CR (1977) Regulation of the immune response. II. Repressor T cells in cyclophosphamide-induced tolerant mice. Eur J Immunol 7:180–185

    Article  PubMed  CAS  Google Scholar 

  10. Yap KL, Ada GL (1978) Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature 273:238–239

    Article  CAS  PubMed  Google Scholar 

  11. Leung KN, Ada GL (1981) Effect of helper cells on the primary in vitro production of delayed-type hypersensitivity to influenza virus. J Exp Med 153:1029–1043

    Article  PubMed  CAS  Google Scholar 

  12. Leung KN, Ashman RB, Ertl HCJ, Ada GL (1980) Selective suppression of the cytotoxic T cell response to influenza infection in mice. Eur J Immunol 10:803–810

    Article  PubMed  CAS  Google Scholar 

  13. Menasche G, Menager MM, Lefebvre JM, Deutsche E, Athman R, Lambert N, Mahlaoui N, Court M, Garin J, Fischer A, de Saint Basile G (2008) A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion. Blood 112:5052–5062

    Article  PubMed  CAS  Google Scholar 

  14. Jenkins MR, Trapani JA, Doherty PC, Turner SJ (2008) Granzyme K expressing cytotoxic T lymphocytes protects against influenza virus in granzyme AB−/− mice. Viral Immunol 21:341–346

    Article  PubMed  CAS  Google Scholar 

  15. Zelinskyy G, Balkow S, Schimmer S, Schepers K, Simon MM, Dittmer U (2004) Independent roles of perforin, granzymes, and Fas in the control of friend retrovirus infection. Virology 330:365–374

    Article  PubMed  CAS  Google Scholar 

  16. He JS, Ostergaard HL (2007) CTLs contain and use intracellular stores of FasL distinct from cytolytic granules. J Immunol 179:2339–2348

    Article  PubMed  CAS  Google Scholar 

  17. Zelinskyy G, Balkow S, Schimmer S, Werner T, Simon MM, Dittmer U (2007) The level of friend retrovirus replication determines the cytolytic pathway of CD8+ T-cell-mediated pathogen control. J Virol 81:11881–11890

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Pascual DW, Wang X, Kochetkova I, Callis G, Richardi C (2008) The absence of lymphoid CD8+ dendritic cell maturation in L-selectin−/− respiratory compartment attenuates antiviral immunity. J Immunol 181:1345–1356

    Article  PubMed  CAS  Google Scholar 

  19. Sato M, Chamoto K, Tsuji T, Iwakura Y, Togashi Y, Koda T, Nishimura T (2001) Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8+ CTL. J Immunol 167:3687–3691

    Article  PubMed  CAS  Google Scholar 

  20. Chattopadhyay S, Chakraborty NG (2005) Continuous presence of Th1 conditions is necessary for longer lasting tumor-specific CTL activity in stimulation cultures with PBL. Hum Immunol 66:884–891

    Article  PubMed  CAS  Google Scholar 

  21. Mailliard RB, Son YI, Redlinger R, Coates PT, Giermasz A, Morel PA, Storkus WJ, Kalinski P (2003) Dendritic cells mediate NK cell help for Th1 and CTL responses: twosignal requirement for the induction of NK cell helper function. J Immunol 171:2366–2373

    Article  PubMed  CAS  Google Scholar 

  22. Chavin KD, Qin L, Yon R, Lin J, Yagita H, Bromberg JS (1994) Anti-CD2 mAbs suppress cytotoxic lymphocyte activity by the generation of Th2 suppressor cells and receptor blockade. J Immunol 152:3729–3739

    PubMed  CAS  Google Scholar 

  23. Terabe M, Park JM, Berzofky JA (2004) Role of IL-13 in regulation of anti-tumor immunity and tumor growth. Cancer Immunol Immunother 53:79–85

    Article  PubMed  CAS  Google Scholar 

  24. Bachuk N, Utermohlen O, Gugel R, Warnecke G, Karow U, Paulsen D, Brombacher F, Kronke M, Deppert W (2007) Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigen-specific CTL in BALB/c mice. Cancer Immunol Immunother 56:1625–1636

    Article  CAS  Google Scholar 

  25. Nishimura M, Sato H, Okazaki H, Satake M, Tadokoro K (2008) Interleukin-10 containing normal human serum inhibits granzyme B release but not perforin release from alloreactive and EBV-specific T cell clones. Cell Immunol 251:31–36

    Article  PubMed  CAS  Google Scholar 

  26. Fernendez MA, Puttur FK, Wang YM, Howden W, Alexander SI, Jones CA (2008) T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice. J Immunol 180:1556–1564

    Article  Google Scholar 

  27. Xu L, Xu W, Jiang Z, Zhang F, Chu Y, Xiong S (2009) Depletion of CD4(+)CD25(high) regulatory T cells from tumor infiltrating lymphocytes predominantly induces Th1 type immune response in vivo which inhibits tumor growth in adoptive immunotherapy. Cancer Biol Ther 8(1):66–72

    Article  PubMed  CAS  Google Scholar 

  28. Chen J, Wang J, Li J, Wu Q, Chu Lim F, Yang P, Hsu HC, Curiel DT, Mountz JD (2008) Enhancement of cytotoxic T-lymphocyte response in aged mice by a novel treatment with recombinant AdIL-12 and wild-type. Adenovirus in rapid succession. Mol Ther 16:1500–1506

    Article  PubMed  CAS  Google Scholar 

  29. Tang F, Zhao LT, Jiang Y, de Ba N, Chi LD, He W (2008) Activity of recombinant human interleukin-15 against tumor recurrence and metastasis in mice. Cell Mol Immunol 5:189–196

    Article  PubMed  PubMed Central  Google Scholar 

  30. Itoh H, Fujimora Y, Tsutsui H, Matsui K, Sugihara A, Terada N, Hada T, Kakishita E, Okamura H, Hara N, Nakanishi K (2004) Involvement of interleukin-18 in acute graft versus-host disease in mice. Transplantation 78:1245–1250

    Article  Google Scholar 

  31. Spolski R, Warren WJ (2008) The Yin and Yang of interleukin-21 in allergy, autoimmunity and cancer. Curr Opin Immunol 20:295–301

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Ansen S, Butler MO, Berezovskaya A, Murray AP, Stevenson K, Nadler LM, Hirano N (2008) Dissociation of its opposing immunologic effects is critical for the optimization of antitumor CD8+ T-cell responses induced by interleukin 21. Clin Cancer Res 14:6125–6136

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Li Y, Yee C (2008) IL-21 mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8+ cytotoxic T lymphocytes. Blood 111:229–235

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Smyth MJ, Hayakawa Y, Creney E, Zerafa N, Sivakumar P, Yagita H, Takeda K (2006) IL-21 enhances tumor-specific CTL induction by anti-DR5 antibody therapy. J Immunol 176:6347–6355

    Article  PubMed  CAS  Google Scholar 

  35. Ha SJ, Kim DJ, Baek KH, Yun YD, Sung YC (2004) IL-23 induces stronger sustained CTL and Th1 immune responses than IL-12 in hepatitis C virus envelope protein 2 DNA immunization. J Immunol 172:525–531

    Article  PubMed  CAS  Google Scholar 

  36. Morishima N, Owaki T, Asakawa M, Kamiya S, Mizuguchi J, Yoshimoto T (2005) Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27. J Immunol 175:1686–1693

    Article  PubMed  CAS  Google Scholar 

  37. Sklavos MM, Tse HM, Piganelli JD (2008) Redox modulation inhibits CD8 T cell effector function. Free Radic Biol Med 45:1477–1486

    Article  PubMed  CAS  Google Scholar 

  38. Bronte V, Serafini P, De Santo C, Marigo I, Tosello V, Mazzoni A, Segal DM, Staib C, Lowel M, Sutter G, Colombo MP, Zanovello P (2003) IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice. J Immunol 170:270–278

    Article  PubMed  CAS  Google Scholar 

  39. Cham CM, Driessens G, O’Keefe JP, Gajewski TF (2008) Glucose deprivation inhibits multiple key gene expression events and effector functions in CD8+ T cells. Eur J Immunol 38:2438–2450

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Ng SP, Silverstone AE, Lai ZW, Zelikoff JT (2006) Effects of prenatal exposure to cigarette smoke on offspring tumor susceptibility and associated immune mechanisms. Toxicol Sci 89:135–144

    Article  PubMed  CAS  Google Scholar 

  41. Hanson CD, Smialowicz RJ (1994) Evaluation of the effect of low-level 2, 3, 7, 8-tetrachlorodibenzo- p-dioxin exposure on cell mediated immunity. Toxicology 88:213–224

    Article  PubMed  CAS  Google Scholar 

  42. Kerkvliet NI, Baecher-Steppan L, Shepard DM, Oughton JA, Dekrey GK (1996) Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2, 3, 7, 8-tetrachlorodibenzo- p-dioxin. J Immunol 157:2310–2319

    PubMed  CAS  Google Scholar 

  43. Lawrence BP, Roberts AD, Neumiller JJ, Cundiff JA, Woodland DL (2006) Aryl hydrocarbon receptor activation impairs the priming but not the recall of influenza virus-specific CD8+ T cells in the lung. J Immunol 177:5819–5828

    Article  PubMed  CAS  Google Scholar 

  44. Li Q, Hirata Y, Piao S, Minani M (2000) Immunotoxicity of N, N-diethylaniline in mice: effect on natural killer activity, cytotoxic T lymphocyte activity, lymphocyte proliferation response and cellular components of the spleen. Toxicology 150:179–189

    Article  PubMed  CAS  Google Scholar 

  45. Henry SP, Levin AA, White K, Mennear JH (2006) Assessment of the effects of ISIS 2302, an anti-sense inhibitor of human ICAM-1, on cellular and Humoral immunity in mice. J Immunotoxicol 3:199–211

    Article  PubMed  CAS  Google Scholar 

  46. De Krey GK, Bauecher-Steppan L, Deyo JA, Smith B, Kerkvliet NI (1993) Polychlorinated biphenyl-induced immune suppression: castration, but not adrenalectomy or RU 38486 treatment, partially restores the suppressed cytotoxic T lymphocyte response to alloantigen. J Pharmacol Exp Ther 267:308–315

    PubMed  Google Scholar 

  47. Karrow NA, McCay JA, Brown R, Musgrove D, Munson AE, White KL Jr (2000) Oxymetholone modulates cell-mediated immunity in male B6C3F1 mice. Drug Chem Toxicol 23:621–644

    Article  PubMed  CAS  Google Scholar 

  48. Sheil JM, Frankenberry MA, Schell TD, Brundage KM, Barnett JB (2006) Propanil exposure induces delayed but sustained abrogation of cell-mediated immunity through direct interference with cytotoxic T-lymphocyte effectors. Environ Health Perspect 114:1059–1064

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Blaylock BL, Soderberg LS, Gandy J, Menna JH, Denton R, Barnett JB (1990) Cytotoxic T-lymphocyte and NK responses in mice treated prenatally with chlordane. Toxicol Lett 51:41–49

    Article  PubMed  CAS  Google Scholar 

  50. Yamada A, Kataoka T, Nagai K (2000) The fungal metabolite gliotoxin: immunosuppressive activity on CTL-mediated cytotoxicity. Immunol Lett 71:27–32

    Article  PubMed  CAS  Google Scholar 

  51. Loser K, Apelt J, Voskort M, Mohaupt M, Balkow S, Schwarz T, Grabbe S, Beissert S (2007) IL-10 controls ultraviolet-induced carcinogenesis in mice. J Immunol 179:365–371

    Article  PubMed  CAS  Google Scholar 

  52. Mumprecht S, Matter M, Pavelic V, Ochsenbein AF (2006) Imatinib mesylate selectively impairs expansion of memory cytotoxic T cells without affecting the control of primary viral infections. Blood 108:3406–3413

    Article  PubMed  CAS  Google Scholar 

  53. Ehrlich JP, Gunnison AF, Burleson GR (1989) Influenza virus-specific cytotoxic T-lymphocyte activity in Fischer 344 rat lungs as a method to assess pulmonary immunocompetence: effect of phosgene inhalation. Inhal Toxicol 1:129–138

    Article  Google Scholar 

  54. Luster MI, Portier C, Pait DG, White KL Jr, Gennings C, Munson AE, Rosenthal GJ (1992) Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam Appl Toxicol 18:200–210

    Article  PubMed  CAS  Google Scholar 

  55. Burleson GR, Burleson FG (2007) Influenza virus host resistance model. Methods 41:31–37

    Article  CAS  PubMed  Google Scholar 

  56. Dietert RR (2008) Influenza host resistance assay to assess developmental immunotoxicity (DIT). Int. J Immunotoxicol 27(6):416

    Google Scholar 

  57. Burleson GR, Burleson FG (2008) Testing human biologicals in animal host resistance models. J Immunotoxicol 5:23–31

    Article  PubMed  Google Scholar 

  58. Burleson GR, Burleson FG (2008) Host resistance models to evaluate immunotoxicity. In: Herzyk D, Bussiere J (eds) Immunotoxicology strategies for pharmaceutical safety assessment. Wiley, Hoboken, NJ

    Google Scholar 

Download references

Acknowledgments

The authors thank Janice Dietert for her editorial suggestions. It is noted that R.R. Dietert previously served as a consultant in developmental immunotoxicology for Burleson Research Technologies, Inc.

Author information

Authors and Affiliations

  1. Burleson Research Technologies, Inc. (BRT), Morrisville, NC, USA

    Gary R. Burleson & Florence G. Burleson

  2. Department of Microbiology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

    Rodney R. Dietert

Authors
  1. Gary R. Burleson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Florence G. Burleson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rodney R. Dietert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gary R. Burleson .

Editor information

Editors and Affiliations

  1. Department of Pharmacology and Toxicology, East Carolina University, Greenville, North Carolina, USA

    Jamie C. DeWitt

  2. Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA

    Cheryl E. Rockwell

  3. Durham, North Carolina, USA

    Christal C. Bowman

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Burleson, G.R., Burleson, F.G., Dietert, R.R. (2018). Evaluation of Cell-Mediated Immune Function Using the Cytotoxic T-Lymphocyte Assay. In: DeWitt, J., Rockwell, C., Bowman, C. (eds) Immunotoxicity Testing. Methods in Molecular Biology, vol 1803. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8549-4_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8549-4_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8548-7

  • Online ISBN: 978-1-4939-8549-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation