Virus-specific T cells

Fast and fully automated generation of virus-specific CD4+ and CD8+ T cells (VSTs)

  • Isolation of virus-specific T cells within 12 h total time and minimal hands-on time
  • Highly convenient and standardized process in a closed and sterile system
  • Easy generation of single- and multi-virus specific CD4+ and CD8+ T cells

Automatic isolation of antigen-specific T cells for adoptive T cell therapy

CD4+ and CD8+ T cells stimulated in vitro with a specific antigen can be magnetically labeled with the CliniMACS® Cytokine Capture System (IFN-gamma). In combination with the new CliniMACS Prodigy, the system allows fast and fully automated generation of antigen-specific T cells in a closed sterile system.

Viral or fungal infections are a major cause of morbidity and mortality in the period of immune recovery after hematopoietic stem cell transplantation (HSCT)1,2. Adoptively transferred antigen-specific T cells have been shown to restore protective immunity and control established adenovirus (AdV)3,4 cytomegalovirus (CMV)5,6 and Epstein-Barr virus (EBV)7,8 infections after HSCT in adults as well as in children. However, the manufacturing of those rare antigen-specific T cells under GMP-compliant conditions is lengthy and very labor-intensive. The CliniMACS® Cytokine Capture System (CCS) (IFN-gamma) addresses all these challenges and provides the perfect solution! Utilizing the CliniMACS Prodigy® Platform, this method allows the fast and fully automated generation of viable antigen-specific CD4+ and CD8+ T cells, e.g., with specificity for HCMV9–12 or BKV13. The whole process happens in a closed sterile system and only takes approximately 12 hours.

The complete CCS workflow

The CliniMACS Prodigy integrates all critical steps of the CliniMACS Cytokine Capture System (IFN-gamma) from antigen-specific T cell stimulation to magnetic isolation and final formulation within 12 h of total time. Importantly, all cell processing steps are automated ensuring a convenient and highly standardized manufacturing process. 
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Application note
Enrichment of human CD4+ and CD8+ antigen-specific T cells – Cytokine Capture System (IFN-gamma)

This application sheet provides an overview of the required and additional materials needed to perform the CliniMACS Prodigy Cytokine Capture System (IFN-gamma) process. It also provides an overview of the process workflow, the setup of the tubing set and literature references.

Antigen-specific T cell stimulation

The first step of the fully automated CCS process is the stimulation of PBMCs with the antigen of interest, e.g. with MACS GMP PepTivator pp65 (CMV). The virus antigen-specific T cells become activated upon antigen encounter, whereas the virus-unspecific T cells remain unaffected by the stimulation. During the CliniMACS Prodigy manufacturing process, the automated antigen-specific stimulation of T cells is performed with high quality and GMP-compliant reagents.

Stimulation with MACS® GMP PepTivator® Peptide Pools
MACS GMP PepTivator Peptide Pools are overlapping oligopeptides covering the complete sequence of a respective antigen. They provide the following benefits:

  • Efficient in-vitro stimulation of CD4+ and CD8+ T cells
  • Generation of multi-virus specific T cells
  • Available for many virus specificitites including CMV, AdV, EBV, BKV and for tumor antigens like WT-1 and NY-ESO-1

T cell culture with TexMACS™ GMP Medium
TexMACS GMP Medium is an optimized medium, recommended for use in combination with the CliniMACS Cytokine Capture System (IFN-gamma). It enables high cell viability, and consistent culture results under serum-free conditions. Filling in flexible bags allows seamless GMP manufacturing of cellular products. Highest product standards are ensured by quality control (QC) functionality tests on every batch and extensive documentation including certificates of analysis (CoA), product data sheets, and regulatory support document cross reference.

Labeling and enrichment of virus-specific T cells

In the second step, all CD45+ cells are labeled with the CliniMACS Catchmatrix Reagent. This is a bispecific antibody, specific for CD45 (binds on cell surface) as well as for IFN-γ (secreted by the target cells). Upon antigen-specific activation, the target cells start to secrete IFN-γ, which is captured by the CliniMACS Catchmatrix Reagent and thereby bound to the surface of the cytokine-secreting cells.

Afterwards, the cells are incubated with the CliniMACS Enrichment Reagent, a second IFN-γ-specific antibody conjugated to MACS MicroBeads resulting in the magnetic labeling of all IFN-γ+ cells. Finally, these cells are magnetically enriched on a MACS Column within the CliniMACS System.

The CliniMACS Catchmatrix Reagent and the CliniMACS Enrichment Reagent are components of the CliniMACS Cytokine Capture System (IFN-gamma).


  1. Feuchtinger, T. et al. (2008) Clinical grade generation of hexon-specific T cells for adoptive T-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation. J. Immunother. 31: 199–206.
  2. Mackinnon et al. (2008) Adoptive cellular therapy for cytomegalovirus infection following allogeneic stem cell transplantation using virus-specific T cells. Blood Cells Mol. Dis. 40: 63–67.
  3. Feuchtinger, T. et al. (2006) Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation. Br. J. Haematol. 134(1): 64–76.
  4. Feucht, J. et al. (2015) Adoptive T-cell therapy with hexon-specific Th1 cells as a treatment of refractory adenovirus infection after HSCT Blood 125: 1986–94.
  5. Feuchtinger, T. et al.. (2010) Adoptive transfer of pp65-specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation. Blood 116: 4360–7.
  6. Peggs, K. S. et al. (2011) Directly Selected Cytomegalovirus-Reactive Donor T Cells Confer Rapid and Safe Systemic Reconstitution of Virus-Specific Immunity Following Stem Cell Transplantation. Clinical Infectious Diseases 52: 49–57.
  7. Moosmann et al. (2010) Effective and long-term control of EBV PTLD after transfer of peptide-selected T cells. Blood 115: 2960–2970.
  8. Icheva, V. et al. (2013) Adoptive Transfer of Epstein-Barr Virus (EBV) Nuclear Antigen 1–Specific T Cells As Treatment for EBV Reactivation and Lymphoproliferative Disorders After
    Allogeneic Stem-Cell Transplantation. J. clin. Oncol. 31: 39–48.
  9. Bunos et al. (2015) Automated isolation of primary antigen-specific T cells from donor lymphocyte concentrates: results of a feasibility exercise. Vox Sang. DOI: 10.1111/vox.12291
  10. Priesner et al. (2016) Comparative Analysis of Clinical-scale IFN-γ-Positive t-Cell enrichment Using Partially and Fully Integrated Platforms. Frontiers Immunology 7: 393.
  11. Kumaresan et al. (2015) Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System. J. Vis. Exp. (104), e52808, doi:10.3791/52808
  12. Kim et al. (2016) Robust Production of Cytomegalovirus pp65-Specific T cells Using a Fully Automated IFN-γ Cytokine Capture System. Blood 128: 5739.

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