Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patient Samples
2.2. T-Cell Reactivity Testing
2.3. Flow Cytometry
2.4. Lymphapheresis
2.5. SARS-CoV-2 Antigen-Specific T-Cell Production
2.6. Cells and Cloning
2.7. Cytotoxicity Assay
2.8. RNA Extraction and RT-PCR
2.9. ACE-2/Spike Antibody Inhibition Assay
2.10. Statistical Analysis
3. Results
3.1. SARS-CoV-2 Antigen-Specific T-Cell Production and Reactivity
3.2. Transfection Efficiency and Expression of M Protein
3.3. M-Protein-Mediated Specific Cytotoxicity Activity and Antibody Titers
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Antigen | Clone | Fluorochrome | Source | Catalog Number |
---|---|---|---|---|
CD3 | UCHT1 | PE/Cy7 | Biolegend | 351304 |
INF-γ | 45-15 | PE | Miltenyi Biotec | 130-113-493 |
CD4 | OKT4 | APC/Cy7 | BD Pharmingen | 317418 |
CD8 | SK1 | PerCP/Cy5.5 | Biolegend | 344710 |
DAPI | Sigma-Aldrich (Merck) | D9542 |
Oligonucleotides | Sequence (5′→3′) |
---|---|
ProtM_XhoI_F | CCGCTCGAGCGGCCACCATGGCAGATTCCAACGGTAC |
ProtM_KpnI_R | CGGGGTACCCCGTTACTGTACAAGCAAAGCAA |
ProtMseq_F | GTAGGCGTGTACGGTGGGAG |
ProtMseq_R | CAGTCGAGGCTGATCAGCGG |
ProtMq_F | GCCACTCCATGGCACTATT |
ProtMq_F | GTATTGCTGGACACCATCTAGG |
GAPDHq_F | GGTGTGAACCATGAGAAGTATGA |
GAPDHq_R | GAGTCCTTCCACGATACCAAAG |
Before Vaccination | After Vaccination | |||
---|---|---|---|---|
Donor 1 | Donor 2 | Donor 1 | Donor 2 | |
Date of apheresis | 28 May 2020 | 14 December 2020 | 26 March 2021 | 06 April 2021 |
TVP | 6819 mL | 2370 mL | 1981 mL | 2441 mL |
ACD anticoagulant | 684 mL | 230 mL | 180 mL | 203 mL |
Product volume | 138 mL | 36 mL | 40 mL | 45 mL |
TNC in product | 7.75 × 109 | 2.6 × 109 | 2.88 × 109 | 2.49 × 109 |
Hematocrit | 2.9% | 3% | 2.7% | 2.3% |
CE | 43% | 7% | 13% | 7% |
Peripheral Blood T-Cells | SARS-CoV-2 Antigen Specific T-Cells | |||||||
---|---|---|---|---|---|---|---|---|
Before Vaccination | After Vaccination | Before Vaccination | After Vaccination | |||||
CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | |
Donor 1 | 76.27% | 21.47% | 66.5% | 27.44% | 94.27% | 4.8% | 82.9% | 9.35% |
Donor 2 | 49.98% | 45.06% | 40.5% | 55.5% | 69.76% | 27.18% | 45.51% | 49.2% |
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García-Ríos, E.; Leivas, A.; Mancebo, F.J.; Sánchez-Vega, L.; Lanzarot, D.; Aguado, J.M.; Martínez-López, J.; Paciello, M.L.; Pérez-Romero, P. Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19. Biomedicines 2022, 10, 630. https://doi.org/10.3390/biomedicines10030630
García-Ríos E, Leivas A, Mancebo FJ, Sánchez-Vega L, Lanzarot D, Aguado JM, Martínez-López J, Paciello ML, Pérez-Romero P. Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19. Biomedicines. 2022; 10(3):630. https://doi.org/10.3390/biomedicines10030630
Chicago/Turabian StyleGarcía-Ríos, Estéfani, Alejandra Leivas, Francisco J. Mancebo, Laura Sánchez-Vega, Diego Lanzarot, José María Aguado, Joaquín Martínez-López, María Liz Paciello, and Pilar Pérez-Romero. 2022. "Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19" Biomedicines 10, no. 3: 630. https://doi.org/10.3390/biomedicines10030630