Challenges for Integrating Immunotoxicology into the Twenty-First-Century Toxicology Testing Paradigm

  • Peer W. F. Karmaus
  • Agnes L. Karmaus
Part of the Methods in Molecular Biology book series (MIMB, volume 1803)


An emerging emphasis on mechanism-focused and human-relevant alternatives to animal use in toxicology underlies the toxicology testing in the twenty-first-century initiative. Herein we describe in vitro high-throughput screening programs seeking to address this goal, as well as strategies established to integrate assay results to build weight of evidence in support of hazard assessment. Furthermore, we discuss unique challenges facing the application of such alternatives for assessing immunotoxicity given the complexity of immune responses. Addressing these challenges will require the development of novel in vitro assays that evaluate well-characterized biochemical processes involved in immune response to help inform on putative adverse outcomes in vivo.

Key words

Immunotoxicology Tox21 Adverse outcome pathway (AOP) Alternative models 



The authors would like to thank Dr. David Allen for the critical review of this chapter. This work was supported in part by the Society of Toxicology Colgate-Palmolive Postdoctoral Fellowship Award in In Vitro Toxicology to P.W.F.K.


  1. 1.
    National Research Council (2007) Toxicity testing in the 21st century: a vision and a strategy. National Academies Press, Washington, DCGoogle Scholar
  2. 2.
    EC (2006) Regulation (EC) No. 1907/2006 of the European Parliament and of the council of 18 December 2006 concerning the registration, evaluation, authorisation and restriction of chemicals (REACH), establishing a European chemicals agency, amending directive 1999/45/EC and repealing council regulation (EEC) no. 793/93 and commission regulation (EC) no. 1488/94 as well as council directive 76/769/EEC and commission directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. OJ L 396:1–520Google Scholar
  3. 3.
    Ankley GT, Bennett RS, Erickson RJ, Hoff DJ, Hornung MW, Johnson RD, Mount DR, Nichols JW, Russom CL, Schmieder PK, Serrrano JA, Tietge JE, Villeneuve DL (2010) Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ Toxicol Chem 29(3):730–741. CrossRefPubMedGoogle Scholar
  4. 4.
    Tollefsen KE, Scholz S, Cronin MT, Edwards SW, de Knecht J, Crofton K, Garcia-Reyero N, Hartung T, Worth A, Patlewicz G (2014) Applying adverse outcome pathways (AOPs) to support integrated approaches to testing and assessment (IATA). Regul Toxicol Pharmacol 70(3):629–640. CrossRefPubMedGoogle Scholar
  5. 5.
    Attene-Ramos MS, Miller N, Huang R, Michael S, Itkin M, Kavlock RJ, Austin CP, Shinn P, Simeonov A, Tice RR, Xia M (2013) The Tox21 robotic platform for the assessment of environmental chemicals—from vision to reality. Drug Discov Today 18(15–16):716–723. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Dix DJ, Houck KA, Martin MT, Richard AM, Setzer RW, Kavlock RJ (2007) The ToxCast program for prioritizing toxicity testing of environmental chemicals. Toxicol Sci 95(1):5–12. CrossRefPubMedGoogle Scholar
  7. 7.
    Kavlock R, Chandler K, Houck K, Hunter S, Judson R, Kleinstreuer N, Knudsen T, Martin M, Padilla S, Reif D, Richard A, Rotroff D, Sipes N, Dix D (2012) Update on EPA’s ToxCast program: providing high throughput decision support tools for chemical risk management. Chem Res Toxicol 25(7):1287–1302. CrossRefPubMedGoogle Scholar
  8. 8.
    Richard AM, Judson RS, Houck KA, Grulke CM, Volarath P, Thillainadarajah I, Yang C, Rathman J, Martin MT, Wambaugh JF, Knudsen TB, Kancherla J, Mansouri K, Patlewicz G, Williams AJ, Little SB, Crofton KM, Thomas RS (2016) ToxCast chemical landscape: paving the road to 21st century toxicology. Chem Res Toxicol 29(8):1225–1251. CrossRefPubMedGoogle Scholar
  9. 9.
    Filer DL, Kothiya P, Setzer RW, Judson RS, Martin MT (2017) tcpl: the ToxCast pipeline for high-throughput screening data. Bioinformatics 33(4):618–620. CrossRefPubMedGoogle Scholar
  10. 10.
    Judson R, Kavlock R, Martin M, Reif D, Houck K, Knudsen T, Richard A, Tice RR, Whelan M, Xia M, Huang R, Austin C, Daston G, Hartung T, Fowle JR 3rd, Wooge W, Tong W, Dix D (2013) Perspectives on validation of high-throughput assays supporting 21st century toxicity testing. ALTEX 30(1):51–56CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wambaugh JF, Wetmore BA, Pearce R, Strope C, Goldsmith R, Sluka JP, Sedykh A, Tropsha A, Bosgra S, Shah I, Judson R, Thomas RS, Setzer RW (2015) Toxicokinetic triage for environmental chemicals. Toxicol Sci 147(1):55–67. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wetmore BA, Wambaugh JF, Allen B, Ferguson SS, Sochaski MA, Setzer RW, Houck KA, Strope CL, Cantwell K, Judson RS, LeCluyse E, Clewell HJ, Thomas RS, Andersen ME (2015) Incorporating high-throughput exposure predictions with dosimetry-adjusted in vitro bioactivity to inform chemical toxicity testing. Toxicol Sci 148(1):121–136. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    OECD (2012) OECD Series on Testing and Assessment No. 168. The adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins. In: OECD Guidelines for the testing of chemicals, Section 4: health effects. OECD Publishing, ParisGoogle Scholar
  14. 14.
    Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R (2011) Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicol In Vitro 25(6):1162–1168. CrossRefPubMedGoogle Scholar
  15. 15.
    Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R (2012) Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol 63(3):489–504. CrossRefPubMedGoogle Scholar
  16. 16.
    Patlewicz G, Kuseva C, Kesova A, Popova I, Zhechev T, Pavlov T, Roberts DW, Mekenyan O (2014) Towards AOP application—implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization. Regul Toxicol Pharmacol 69(3):529–545. CrossRefPubMedGoogle Scholar
  17. 17.
    OECD (2015) Test No. 442C. In chemico skin sensitisation: Direct Peptide Reactivity Assay (DPRA). In: OECD Guidelines for the testing of chemicals, Section 4: health effects. OECD Publishing, ParisGoogle Scholar
  18. 18.
    OECD (2015) Test No. 442D. in vitro skin sensitisation: ARE-Nrf2 Luciferase test method. In: OECD Guidelines for the testing of chemicals, Section 4: Health effects. OECD Publishing, ParisGoogle Scholar
  19. 19.
    OECD (2016) Test No. 442E: in vitro skin sensitisation: human cell line activation test (h-CLAT). In: OECD Guidelines for the testing of chemicals, Section 4: health effects. OECD Publishing, ParisGoogle Scholar
  20. 20.
    OECD (2010) Test No. 429: skin sensitisation: local lymph node assay. In: OECD Guidelines for the testing of chemicals, Section 4: health effects. OECD Publishing, ParisGoogle Scholar
  21. 21.
    Strickland J, Zang Q, Paris M, Lehmann DM, Allen D, Choksi N, Matheson J, Jacobs A, Casey W, Kleinstreuer N (2016) Multivariate models for prediction of human skin sensitization hazard. J Appl Toxicol 37(3):347–360. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Zang Q, Paris M, Lehmann DM, Bell S, Kleinstreuer N, Allen D, Matheson J, Jacobs A, Casey W, Strickland J (2017) Prediction of skin sensitization potency using machine learning approaches. J Appl Toxicol 37(7):792–805. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    JRC-EU (2013) EURL ECVAM recommendation on the Direct Peptide Reactivity Assay (DPRA) for skin sensitisation testing. Publications Office of the European Union, LuxembourgGoogle Scholar
  24. 24.
    JRC-EU (2014) EURL ECVAM recommendation on the KeratinoSens™ assay for skin sensitisation testing. Publications Office of the European Union, LuxembourgGoogle Scholar
  25. 25.
    JRC-EU (2015) EURL ECVAM recommendation on the human cell line activation test (h-CLAT) for skin sensitization testing. Publications Office of the European Union, LuxembourgGoogle Scholar
  26. 26.
    DuPage M, Bluestone JA (2016) Harnessing the plasticity of CD4(+) T cells to treat immune-mediated disease. Nat Rev Immunol 16(3):149–163. CrossRefPubMedGoogle Scholar
  27. 27.
    Li J, Lu E, Yi T, Cyster JG (2016) EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells. Nature 533(7601):110–114. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Crotty S (2014) T follicular helper cell differentiation, function, and roles in disease. Immunity 41(4):529–542. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lu KT, Kanno Y, Cannons JL, Handon R, Bible P, Elkahloun AG, Anderson SM, Wei L, Sun H, O’Shea JJ, Schwartzberg PL (2011) Functional and epigenetic studies reveal multistep differentiation and plasticity of in vitro-generated and in vivo-derived follicular T helper cells. Immunity 35(4):622–632. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Vinuesa CG, Linterman MA, Yu D, MacLennan IC (2016) Follicular helper T cells. Annu Rev Immunol 34:335–368. CrossRefPubMedGoogle Scholar
  31. 31.
    Artis D, Spits H (2015) The biology of innate lymphoid cells. Nature 517(7534):293–301. CrossRefPubMedGoogle Scholar
  32. 32.
    Kaplan BL, Li J, LaPres JJ, Pruett SB, Karmaus PW (2015) Contributions of nonhematopoietic cells and mediators to immune responses: implications for immunotoxicology. Toxicol Sci 145(2):214–232. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Malhotra D, Fletcher AL, Turley SJ (2013) Stromal and hematopoietic cells in secondary lymphoid organs: partners in immunity. Immunol Rev 251(1):160–176. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Mueller SN, Germain RN (2009) Stromal cell contributions to the homeostasis and functionality of the immune system. Nat Rev Immunol 9(9):618–629. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Roozendaal R, Mebius RE (2011) Stromal cell-immune cell interactions. Annu Rev Immunol 29:23–43. CrossRefPubMedGoogle Scholar
  36. 36.
    Hammad H, Lambrecht BN (2008) Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat Rev Immunol 8(3):193–204. CrossRefPubMedGoogle Scholar
  37. 37.
    Li F, Tian Z (2013) The liver works as a school to educate regulatory immune cells. Cell Mol Immunol 10(4):292–302. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Peterson LW, Artis D (2014) Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 14(3):141–153. CrossRefPubMedGoogle Scholar
  39. 39.
    Sternberg EM (2006) Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat Rev Immunol 6(4):318–328. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Girard JP, Moussion C, Forster R (2012) HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes. Nat Rev Immunol 12(11):762–773. CrossRefPubMedGoogle Scholar
  41. 41.
    Buck MD, O'Sullivan D, Pearce EL (2015) T cell metabolism drives immunity. J Exp Med 212(9):1345–1360. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Ganeshan K, Chawla A (2014) Metabolic regulation of immune responses. Annu Rev Immunol 32:609–634. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kelly B, O’Neill LA (2015) Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res 25(7):771–784. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    MacIver NJ, Michalek RD, Rathmell JC (2013) Metabolic regulation of T lymphocytes. Annu Rev Immunol 31:259–283. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Netea MG, Quintin J, van der Meer JW (2011) Trained immunity: a memory for innate host defense. Cell Host Microbe 9(5):355–361. CrossRefPubMedGoogle Scholar
  46. 46.
    Chi H (2012) Regulation and function of mTOR signalling in T cell fate decisions. Nat Rev Immunol 12(5):325–338. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Weichhart T, Hengstschlager M, Linke M (2015) Regulation of innate immune cell function by mTOR. Nat Rev Immunol 15(10):599–614. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Yao Y, Liu R, Shin MS, Trentalange M, Allore H, Nassar A, Kang I, Pober JS, Montgomery RR (2014) CyTOF supports efficient detection of immune cell subsets from small samples. J Immunol Methods 415:1–5. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of ImmunologySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Integrated Laboratory Systems, Inc.Research Triangle ParkUSA

Personalised recommendations