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

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Abstract

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.

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About the authors

I. A. Ishina

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

Author for correspondence.
Email: ishina.irina.a@gmail.com
Russian Federation, Moscow

M. Y. Zakharova

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

Email: mariya.zakharova333@gmail.com
Russian Federation, Moscow

I. N. Kurbatskaia

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

Email: ishina.irina.a@gmail.com
Russian Federation, Moscow

A. E. Mamedov

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

Email: ishina.irina.a@gmail.com
Russian Federation, Moscow

A. 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

Russian Federation, Moscow; Moscow

Y. P. Rubtsov

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

Email: ishina.irina.a@gmail.com
Russian Federation, Moscow

A. G. 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

Russian Federation, Moscow; Moscow; Moscow

References

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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.

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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.

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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.

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