Original Article

The Effect of Antigen Dose and Antigen Presenting Process on T Cell Stimulation: A Method for Enrichment of TB10.4 Antigen-specific T-cell Clones


T-lymphocytes have critical functions in the immune responses against viral and intracellular bacterial infections as well as cancers. Antigen (Ag)-specific T-lymphocyte clones enriched and expanded in vitro are valuable tools in the study of immune responses in animal models and adoptive T-cell therapy of patients with cancer or infection.
We described a method for inducing, enriching, and replicating Ag-specific poly-clonal T-cells from BALB/c mice infected with live Bacillus Calmette Guérin (BCG) bacterium. During a 7-8 days procedure, T-lymphocytes were purified from immune cells of lymph nodes stimulated with immunodominant Ag of BCG, TB10.4, and expanded by interleukin -2 cytokine. We evaluated the effect of Ag doses (1, 10, and 100 μg/mL) and exposure method of Ag presenting cells (APCs) to T-cells, on T-cells’ proliferation, viability, and Interferon-gamma (IFN-γ) secretion at 2, 5, and 7 days after Ag stimulation.
Increasing Ag concentration increased the average cell division, but at the highest dose of Ag (100 μg/mL), T-cell viability is decreased. Only clones induced by 10 μg/mL Ag produced a desirable amount of IFN-γ. Incubation of Ag and APCs, 24 h before T-lymphocytes addition, increased the proliferation and viability of cells. T cells are in a more favorable condition around day 5 of Ag stimulation in terms of proliferation and survival, and it is the desired time for T cell restimulation.
For optimal preparation of specific T-cells for adoptive cell transfer, optimization of Ag dose, the order of APCs and T-cells exposure with Ag, and the duration of initial Ag stimulation, as well as the time for restimulation, is essential.

1. Adams NM, Grassmann S, Sun JC. Clonal expansion of innate and adaptive lymphocytes. Nat Rev Immunol. 2020;20(11):694-707.
2. Messaoudi I, Patino JAG, Dyall R, LeMaoult J, Nikolich-Žugich J. Direct link between mhc polymorphism, T cell avidity, and diversity in immune defense. Science. 2002;298(5599):1797-800.
3. Speiser DE, Liénard D, Pittet MJ, Batard P, Rimoldi D, Guillaume P, et al. In vivo activation of melanoma-specific CD8+ T cells by endogenous tumor antigen and peptide vaccines. A comparison to virus-specific T cells. Eur J Immunol. 2002;32(3):731-41.
4. Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol. 2020:1-18.
5. Yee C. Adoptive Therapy Using Antigen-Specific T-Cell Clones. Cancer J. 2010;16(4):367-73.
6. Wei J, Han X, Bo J, Han W. Target selection for CAR-T therapy. J Hematol Oncol. 2019;12(1):1-9.
7. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. 2011;17(13):4550-7.
8. Heslop HE, Slobod KS, Pule MA, Hale GA, Rousseau A, Smith CA, et al. Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients. Blood. 2010;115(5):925-35.
9. Rothoeft T, Gonschorek A, Bartz H, Anhenn O, Schauer U. Antigen dose, type of antigen-presenting cell and time of differentiation contribute to the T helper 1/T helper 2 polarization of naive T cells. Immunology. 2003;110(4):430-9.
10. Nikolich-Žugich J, Slifka MK, Messaoudi I. The many important facets of T-cell repertoire diversity. Nat Rev Immunol. 2004;4(2):123-32.
11. Labarrière N, Fortun A, Bellec A, Khammari A, Dreno B, Saiagh S, et al. A full GMP process to select and amplify epitope-specific T lymphocytes for adoptive immunotherapy of metastatic melanoma. Clin Dev Immunol. 2013;2013.
12. Lim JF, Berger H, Su IH. Isolation and Activation of Murine Lymphocytes. J Vis Exp. 2016(116):54596.
13. Hensel JA, Khattar V, Ashton R, Ponnazhagan S. Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations. Lab Invest. 2019;99(1):93-106.
14. Hervas-Stubbs S, Majlessi L, Simsova M, Morova J, Rojas M-J, Nouzé C, et al. High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection. Infect Immun. 2006;74(6):3396-407.
15. Skjøt RLV, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S, et al. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as Immunodominant T-cell antigens. Infect Immun. 2000;68(1):214-20.
16. Skjøt RLV, Brock I, Arend SM, Munk ME, Theisen M, Ottenhoff TH, et al. Epitope mapping of the immunodominant antigen TB10. 4 and the two homologous proteins TB10. 3 and TB12. 9, which constitute a subfamily of the esat-6 gene family. Infect Immun. 2002;70(10):5446-53.
17. Gholoobi A, Sankian M, Zarif R, Farshadzadeh Z, Youssefi F, Sadeghian A, et al. Molecular cloning, expression and purification of protein TB10. 4 secreted by mycobacterium tuberculosis. Ira J Med Sciences. 2010;13(4):189-93.
18. Hathcock KS. T cell enrichment by nonadherence to nylon. Curr Protoc Immunol. 1999;30(1):3.2. 1-3.2. 4.
19. Zanganeh E, Soudi S, Zavaran Hosseini A, Khosrojerdi A. Repeated intravenous injection of adipose tissue derived mesenchymal stem cells enhances Th1 immune responses in Leishmania major-infected BALB/c mice. Immunol Lett. 2019;216:97-105.
20. Neller MA, Lai MH-L, Lanagan CM, Linda E, Pritchard AL, Martinez NR, et al. High efficiency ex vivo cloning of antigen-specific human effector T cells. PloS one. 2014;9(11):e110741.
21. Van Heijst JWJ, Gerlach C, Swart E, Sie D, Nunes-Alves C, Kerkhoven RM, et al. Recruitment of Antigen-Specific CD8+ T Cells in Response to Infection Is Markedly Efficient. Science. 2009;325(5945):1265-9.
22. Kim M, Moon H-B, Kim K, Lee K-Y. Antigen dose governs the shaping of CTL repertoires in vitro and in vivo. Int Immunol. 2006;18(3):435-44.
23. Henrickson SE, Mempel TR, Mazo IB, Liu B, Artyomov MN, Zheng H, et al. T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation. Nat Immunol. 2008;9(3):282-91.
24. Ivashkiv LB. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat Rev Immunol. 2018;18(9):545-58.
25. Hosken NA, Shibuya K, Heath AW, Murphy KM, O'Garra A. The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model. J Exp Med. 1995;182(5):1579-84.
26. Critchfield J, Racke M, Zuniga-Pflucker J, Cannella B, Raine C, Goverman J, et al. T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis. Science. 1994;263(5150):1139-43.
27. Iezzi G, Karjalainen K, Lanzavecchia A. The Duration of Antigenic Stimulation Determines the Fate of Naive and Effector T Cells. Immunity. 1998;8(1):89-95.
28. Meier S, Stark R, Frentsch M, Thiel A. The influence of different stimulation conditions on the assessment of antigen-induced CD154 expression on CD4+ T cells. Cytometry A. 2008;73A(11):1035-42.
29. Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, Wieczorek G, et al. Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol. 2009;39(4):1088-97.
IssueVol 20 No 3 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijaai.v20i3.6338
Antigens Antigen-presenting cells Cell- and tissue-based therapy Clone cells Immunotherapy T-lymphocytes

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Motiee M, Zavaran Hosseini A, Soudi S, Hassanzadeh SM. The Effect of Antigen Dose and Antigen Presenting Process on T Cell Stimulation: A Method for Enrichment of TB10.4 Antigen-specific T-cell Clones. Iran J Allergy Asthma Immunol. 2021;20(3):364-375.