Antigenic peptide–thioredoxin fusion chimeras for in vitro stimulus of CD4+ TCR+ Jurkat T-cells

Мұқаба

Дәйексөз келтіру

Толық мәтін

Ашық рұқсат Ашық рұқсат
Рұқсат жабық Рұқсат берілді
Рұқсат жабық Тек жазылушылар үшін

Аннотация

Study of CD4+ T-cell response and T-cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T-cell assays may be a critical point in such studies. Here we present a T-cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP-cells with CD80+ HLA-DRB1*01:01+ HeLa-cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa-cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T-cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.

Толық мәтін

Рұқсат жабық

Авторлар туралы

I. Ishina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Хат алмасуға жауапты Автор.
Email: ishina.irina.a@gmail.com
Ресей, Moscow

M. Zakharova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: mariya.zakharova333@gmail.com
Ресей, Moscow

I. Kurbatskaia

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ishina.irina.a@gmail.com
Ресей, Moscow

A. Mamedov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ishina.irina.a@gmail.com
Ресей, Moscow

A. Belogurov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Evdokimov Moscow State University of Medicine and Dentistry

Email: ishina.irina.a@gmail.com

Department of Biological Chemistry

Ресей, Moscow; Moscow

Y. Rubtsov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ishina.irina.a@gmail.com
Ресей, Moscow

A. Gabibov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Higher School of Economics; Lomonosov Moscow State University

Email: ishina.irina.a@gmail.com

Academician of the RAS, Department of Life Sciences, Department of Chemistry

Ресей, Moscow; Moscow; Moscow

Әдебиет тізімі

  1. Pishesha N., Harmand T.J., Ploegh H.L. A Guide to Antigen Processing and Presentation // Nat. Rev. Immunol. 2022. V. 22. P. 751–764.
  2. Santambrogio L. Molecular Determinants Regulating the Plasticity of the MHC Class II Immunopeptidome // Front. Immunol. 2022. V. 13.
  3. Ishina I.A., Zakharova M.Y., Kurbatskaia I.N., et al. MHC Class II Presentation in Autoimmunity // Cells. 2023. V. 12.
  4. Chen B., Khodadoust M.S., Olsson N., et al. Predicting HLA Class II Antigen Presentation through Integrated Deep Learning // Nat. Biotechnol. 2019. V. 37. P. 1332–1343.
  5. Racle J., Guillaume P., Schmidt J., et al. Machine Learning Predictions of MHC-II Specificities Reveal Alternative Binding Mode of Class II Epitopes // Immunity. 2023. V. 56. P. 1359–1375.
  6. Butler M.O., Ansén S., Tanaka M., et al. A Panel of Human Cell-based Artificial APC Enables the Expansion of Long-lived Antigen-specific CD4+ T Cells Restricted by Prevalent HLA-DR Alleles // Int. Immunol. 2010. V. 22. P. 863– 873.
  7. Garnier A., Hamieh M., Drouet A., et al. Artificial Antigen-presenting Cells Expressing HLA Class II Molecules as an Effective Tool for Amplifying Human Specific Memory CD4+ T Cells // Immunol. Cell. Biol. 2016. V. 94. P. 662–672.
  8. Ishina I.A., Kurbatskaia I.N., Mamedov A.E., et al. Genetically Engineered CD80–pMHC-harboring Extracellular Vesicles for Antigen-specific CD4+ T-cell Engagement // Front. Bioeng. Biotechnol. 2024. V. 11.
  9. Rosskopf S., Leitner J., Paster W., et al. A Jurkat 76 Based Triple Parameter Reporter System to Evaluate TCR Functions and Adoptive T Cell Strategies // Oncotarget. 2018. V. 9. 17608.
  10. Hennecke J., Carfi A., Wiley D. C. Structure of a Covalently Stabilized Complex of a Human αβ T-cell Receptor, Influenza HA Peptide and MHC Class II Molecule, HLA-DR1 // EMBO J. 2000. V. 19. P. 5611–5624.
  11. Hahn M., Nicholson M.J., Pyrdol J., Wucherpfennig K.W. Unconventional Topology of Self Peptide–Major Histocompatibility Complex Binding by a Human Autoimmune T Cell Receptor // Nat. Immunol. 2005. V. 6. P. 490–496.
  12. Belogurov A.A., Kurkova I.N., Friboulet A., et al. Recognition and Degradation of Myelin Basic Protein Peptides by Serum Autoantibodies: Novel Biomarker for Multiple Sclerosis // The Journal of Immunology. 2008. V. 180. P. 1258–1267.
  13. Mamedov A., Vorobyeva N., Filimonova I., et al. Protective Allele for Multiple Sclerosis HLA-DRB1*01:01 Provides Kinetic Discrimination of Myelin and Exogenous Antigenic Peptides // Front. Immunol. 2020. V. 10.
  14. Álvaro-Benito M., Freund C. Revisiting Nonclassical HLA II Functions in Antigen Presentation: Peptide Editing and Its Modulation // HLA. 2020. V. 96. P. 415–429.

Қосымша файлдар

Қосымша файлдар
Әрекет
1. JATS XML
Жүктеу
2. Fig. 1. Schematic representation of the use of trx peptides to activate CD4+ TCR+ Jurkat 76 TPR cells. Trx peptides were expressed in E. coli and purified with Ni-NTA. The resulting protein was loaded onto HLA-DRB1*01:01 or HLA-DRB1*15:01 CD80+ HeLa cell lines and incubated with the corresponding CD4+ TCR+ Jurkat 76 TPR cell line. Subsequent activation of the CD4+ TCR+ Jurkat 76 TPR cell line occurred through the formation of a trimolecular complex and was assessed by GFP expression induced by NFAT activation.

Жүктеу (151KB)
Метадеректер
3. Fig. 2. Stimulation of CD4+ HA1.7+ TCR Jurkat 76 TPR by CD80+ HLA-DRB1*01:01+ HeLa cells primed with MHC-II epitopes. CD4+ HA1.7 TCR+ Jurkat 76 TPR cells were incubated with CD80+ HLA-DRB1*01:01+ HeLa for 16 h with synthetic HA peptide or trx-HA at concentrations of 0.5, 1, 5, 10, and 20 μM. trx vehicle was used as a negative control. Analysis was performed by flow cytometry. Values ​​indicate the percentage of activated cells expressing GFP. Representative flow cytometry profiles are shown. The percentage of GFP-positive CD4+ HA1.7 TCR+ Jurkat 76 TPR cell lines incubated with CD80+ HLA-DRB1*01:01+ HeLa cells loaded with synthetic HA peptide or trx-HA at concentrations of 0.5, 1, 5, 10 and 20 μM are shown as the mean ± standard deviation of three replicates (lower panel). Statistical analysis was performed using Welch's t-test: ** – p < 0.01, *** – p < 0.001.

Жүктеу (284KB)
Метадеректер
4. Fig. 3. Stimulation of CD4+ Ob.1A12 TCR+ Jurkat 76 TPR cells by CD80+ HLA-DRB1*15:01+ HeLa cells primed with MHC-II epitopes. CD4+ Ob.1A12 TCR+ Jurkat 76 TPR cells were incubated with CD80+ HLA-DRB1*15:01+ HeLa cells for 16 h with synthetic MBP or trx-MBP peptide at concentrations of 0.5, 1, 5, 10, and 20 μM. trx vehicle was used as a negative control. Analysis was performed by flow cytometry. Values ​​indicate the percentage of activated cells expressing GFP. Representative flow cytometry profiles are shown. The percentage of GFP-positive CD4+ Ob.1A12 TCR+ Jurkat 76 TPR cell lines incubated with CD80+ HLA-DRB1*15:01+ HeLa loaded with synthetic MBP or trx-MBP peptide at concentrations of 0.5, 1, 5, 10 and 20 μM are shown as the mean ± standard deviation of three experimental replicates (lower panel). Statistical analysis was performed using Welch's t-test: * – p < 0.05, ** – p < 0.01, *** – p < 0.001.

Жүктеу (281KB)
Метадеректер

© Russian Academy of Sciences, 2024