Generation of T. cruzi-Specific Primary CD4+ T Cell Lines from Peripheral Blood Mononuclear Cells Isolated from Chagas Disease Patients

  • Gonzalo R. AcevedoEmail author
  • Paula B. Alcaráz
  • Clemencia Pinilla
  • Karina A. Gómez
Part of the Methods in Molecular Biology book series (MIMB, volume 1955)


Human CD8+ and CD4+ T cell lines and clones are valuable tools to explore the role of these cells in the context of diseases, especially in cases in which the main underlying actor is the immune response, like Chagas disease. These cell lines and clones provide a good experimental system to address the phenotypic and functional features of specific T cell subpopulations and furthermore settle the framework necessary for analyzing their antigen/peptide specificity.

This chapter details a culture method for the establishment of T. cruzi-specific memory T cell lines from mononuclear cells isolated from Chagas disease patients’ peripheral blood. The presented protocol comprises (1) enrichment of memory CD4+ T cells, (2) stimulation with parasite lysate, (3) evaluation of specificity, and (4) expansion and maintenance of specific T cell lines.

Key words

Antigen-specific T cells T cell line Memory C4 T cells Trypanosoma cruzi Chagas disease 



The funds that permitted the development of the method hereby described were granted by the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 112-200801-02915), the Agencia Nacional de Promoción Científica y Tecnológica (PICT2014-1026), the AJ Roemmers Foundation (Clinical Research Grant for Young Investigators 2016–2018), and the National Institute of Allergy and Infectious Diseases (5R21AI109439-02).


  1. 1.
    Junqueira C, Caetano B, Bartholomeu DC, Melo MB, Ropert C, Rodrigues MM, Gazzinelli RT (2010) The endless race between Trypanosoma cruzi and host immunity: lessons for and beyond Chagas disease. Expert Rev Mol Med 12:e29CrossRefGoogle Scholar
  2. 2.
    Dutra WO, Gollob KJ (2008) Current concepts in immunoregulation and pathology of human Chagas disease. Curr Opin Infect Dis 21:287–292CrossRefGoogle Scholar
  3. 3.
    dos Santos Virgilio F, Pontes C, Dominguez MR et al (2014) CD8+ T cell-mediated immunity during Trypanosoma cruzi infection: a path for vaccine development? Mediat Inflamm 2014:1–12CrossRefGoogle Scholar
  4. 4.
    Ben Younes-Chennoufi A, Said G, Eisen H, Durand A, Hontebeyrie-Joskowicz M (1988) Cellular immunity to Trypanosoma cruzi is mediated by helper T cells (CD4+). Trans R Soc Trop Med Hyg 82:84–89CrossRefGoogle Scholar
  5. 5.
    Tarleton RL, Koller BH, Latour A, Postan M (1992) Susceptibility of β2-microglobulin-deficient mice to Trypanosoma cruzi infection. Nature 356:338–340CrossRefGoogle Scholar
  6. 6.
    Pérez-Molina JA, Molina I (2017) Chagas disease. Lancet 6736:1–13Google Scholar
  7. 7.
    Fiuza JA, Fujiwara RT, Gomes JAS et al (2009) Profile of central and effector memory T cells in the progression of chronic human Chagas disease. PLoS Negl Trop Dis 3:e512. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Mateus J, Lasso P, Pavia P et al (2015) Low frequency of circulating CD8+ T stem cell memory cells in chronic chagasic patients with severe forms of the disease. PLoS Negl Trop Dis 9:e3432CrossRefGoogle Scholar
  9. 9.
    Souza PEA, Rocha MOC, Menezes CAS, Coelho JS, Chaves ACL, Gollob KJ, Dutra WO (2007) Trypanosoma cruzi infection induces differential modulation of costimulatory molecules and cytokines by monocytes and T cells from patients with indeterminate and cardiac Chagas’ disease. Infect Immun 75:1886–1894CrossRefGoogle Scholar
  10. 10.
    Argüello RJ, Albareda MC, Alvarez MG, Bertocchi G, Armenti AH, Vigliano C, Meckert PC, Tarleton RL, Laucella SA (2012) Inhibitory receptors are expressed by Trypanosoma cruzi-specific effector T cells and in hearts of subjects with chronic Chagas disease. PLoS One 7:e35966. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Silva J, Morrisey P, Grabstein K, Mohler K, Anderson D, Reed S (1992) Interleukin 10 and interferon γ regulation of experimental Trypanosoma cruzi infection. J Exp Med 175:169–174CrossRefGoogle Scholar
  12. 12.
    Vitelli-Avelar DM, Sathler-Avelar R, Dias JCP, Pascoal VPM, Teixeira-Carvalho A, Lage PS, Elói-Santos SM, Corrêa-Oliveira R, Martins-Filho OA (2005) Chagasic patients with indeterminate clinical form of the disease have high frequencies of circulating CD3 +CD16 -CD56 + natural killer T cells and CD4 +CD25 high regulatory T lymphocytes. Scand J Immunol 62:297–308CrossRefGoogle Scholar
  13. 13.
    Laucella SA, Riarte A, Prado N, Zapata J, Segura EL (2001) α4 integrins and sialyl Lewis x modulation in chronic Chagas disease: further evidence of persistent immune activation. Scand J Immunol 53:514–519CrossRefGoogle Scholar
  14. 14.
    Petray PB, Rottenberg ME, Bertot G, Corral RS, Diaz A, Örn A, Grinstein S (1993) Effect of anti-γ-interferon and anti-interleukin-4 administration on the resistance of mice against infection with reticulotropic and myotropic strains of Trypanosoma cruzi. Immunol Lett 35:77–80CrossRefGoogle Scholar
  15. 15.
    Rodrigues MM, Ribeirão M, Pereira-Chioccola V, Renia L, Costa F (1999) Predominance of CD4 Th1 and CD8 Tc1 cells revealed by characterization of the cellular immune response generated by immunization with a DNA vaccine containing a Trypanosoma cruzi gene. Infect Immun 67:3855–3863PubMedPubMedCentralGoogle Scholar
  16. 16.
    Albareda MC, Olivera GC, Laucella S a, Alvarez MG, Fernandez ER, Lococo B, Viotti R, Tarleton RL, Postan M (2009) Chronic human infection with Trypanosoma cruzi drives CD4+ T cells to immune senescence. J Immunol 183:4103–4108CrossRefGoogle Scholar
  17. 17.
    Longhi SA, Atienza A, Prados GP et al (2014) Cytokine production but lack of proliferation in peripheral blood mononuclear cells from chronic Chagas’ disease cardiomyopathy patients in response to T. cruzi ribosomal P proteins. PLoS Negl Trop Dis 8:e2906. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Magalhães LMDD, Villani FNAA, Nunes M do CP, Gollob KJ, Rocha MOCC, Dutra WO (2013) High interleukin 17 expression is correlated with better cardiac function in human Chagas disease. J Infect Dis 207:661–665CrossRefGoogle Scholar
  19. 19.
    Guedes PMM, Gutierrez FRS, Silva GK et al (2012) Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas’ disease. PLoS Negl Trop Dis 6:e1630. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Sousa GR, Gomes JAS, Damasio MPS, Nunes MCP, Costa HS, Medeiros NI, Fares RCG, Chaves AT, Corrêa-Oliveira R, Rocha MOC (2017) The role of interleukin 17-mediated immune response in Chagas disease: high level is correlated with better left ventricular function. PLoS One 12:e0172833CrossRefGoogle Scholar
  21. 21.
    Rodríguez-Angulo H, Marques J, Mendoza I, Villegas M, Mijares A, Gironès N, Fresno M (2017) Differential cytokine profiling in Chagasic patients according to their arrhythmogenic-status. BMC Infect Dis 17:1–10CrossRefGoogle Scholar
  22. 22.
    Acevedo GR, Girard MC, Gómez KA (2018) The unsolved jigsaw puzzle of the immune response in Chagas disease. Front Immunol 9:1929. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Acevedo GR, Longhi SA, Bunying A, Sabri N, Zago P, Santos R, Judkowski VA, Atienza A (2017) Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients. PLoS One 12:e0178380CrossRefGoogle Scholar
  24. 24.
    Tosato G, Cohen JI (2007) Generation of Epstein-Barr virus (EBV)-immortalized B cell lines. Curr Protoc Immunol Chapter 7:Unit 7.22Google Scholar
  25. 25.
    Ripoll JG, Giraldo NA, Bolaños NI, Roa N, Rosas F, Cuéllar A, Puerta CJ, González JM (2017) T cells responding to Trypanosoma cruzi detected by membrane TNF-α and CD154 in chagasic patients. Immun Inflamm Dis 6:1–11Google Scholar
  26. 26.
    Judkowski VA, Santos RG, Acevedo G, Giulianotti MA, Appel JR, Longhi S, Gomez KA, Pinilla C (2015) Antigen discovery for the identification of vaccine candidates and biomarkers using a T cell driven approach in combination with positional scanning peptide libraries. Procedia Vaccinol 9:91–95CrossRefGoogle Scholar
  27. 27.
    Pinilla C, Appel JR, Judkowski V, Houghten RA (2012) Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries. Curr Protoc Immunol.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Gonzalo R. Acevedo
    • 1
    Email author
  • Paula B. Alcaráz
    • 1
  • Clemencia Pinilla
    • 2
  • Karina A. Gómez
    • 1
  1. 1.Laboratorio de Inmunología de las Infecciones por TripanosomátidosInstituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Buenos AiresArgentina
  2. 2.Torrey Pines Institute for Molecular Studies (TPIMS)Port St. LucieUSA

Personalised recommendations