Fast and fully automated separation of IFN-gamma secreting CD4+ and CD8+ T cells

  • Restore immunity against, for example, CMV, AdV and EBV
  • Highly convenient and standardized process in a closed and sterile system
  • Easy generation of single- and multi-virus specific CD4+ and CD8+ T cells within twelve hours
Video

Automatic separation of antigen-specific T cells 

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 CliniMACS Prodigy® Instrument, the system allows fast and fully automated separation 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)3,4,13, cytomegalovirus (CMV)5,6, and Epstein-Barr virus (EBV)7,8 infections after HSCT in adults as well as in children.

With the CliniMACS® Cytokine Capture System (CCS) (IFN-gamma) virus-specific T cells can be isolated in a fast and easy process.

Utilizing the CliniMACS Prodigy Platform, this method allows the fully automated and reproducible separation of viable antigen-specific CD4+ and CD8+ T cells, e.g., with specificity for HCMV9–12, EBV14, AdV14, BKV15 or multivirus-specificity14. The whole process is facilitated in a closed sterile system and only takes approximately twelve hours.

The complete CCS workflow

The CliniMACS Prodigy integrates all critical steps of the CCS workflow from antigen-specific T cell stimulation to labeling of target cells, magnetic isolation and final formulation within twelve hours of total time. Importantly, all cell processing steps are automated ensuring a convenient and highly standardized separation process. 

1. Eligibility test of donor blood

Virus-specific T cells can be enriched based on their secretion of IFN-γ after restimulation with the appropriate antigen by using the CCS. As this technology targets existing virus-specific T cells from donor blood materials (e.g. leukapheresis), it is recommended to validate the eligibility of each blood donor before running the CCS. This can be done, for example, with the help of our special protocol (Blood donor eligibility test, research use only), optimized for this test. The associated Express Mode on a MASCSQuant® Flow Cytometer simplifies the analysis and allows for a fully automated and standardized cell analysis. 

2. Antigen-specific T cell stimulation

The second step of the fully automated process on the CliniMACS Prodigy is the stimulation of PBMCs with the antigen of interest. This can be performed, for example, with MACS® GMP PepTivator® Peptide Pools. Their unique structure of overlapping oligopeptides cover the complete sequence of a respective antigen and provides a number of benefits:

·       Efficient in vitro stimulation of CD4+ and CD8+ T cells

·       Targets multiple immunodominant epitopes

·       Available for many virus specificities including CMV, AdV, EBV, BKV and for tumor antigens like WT-1 and NY-ESO-1

3. Cell labeling with CliniMACS Catchmatrix Reagent

In the next step CD45+ cells are being labeled with a bispecific antibody, the CliniMACS Catchmatrix Reagent. On the one hand this antibody tandem is specific for CD45 and binds to the cell surface. On the other hand it is specific for IFN-γ, which is secreted by the stimulated target cells during the secretion period. After secretion, IFN-γ is captured by the CliniMACS Catchmatrix Reagent and thereby bound to the surface of the cytokine-secreting cells.

4. Cell labeling with CliniMACS Enrichment Reagent

The cell surface-bound IFN-γ is targeted by the CliniMACS Enrichment Reagent. This IFN-γ-specific antibody is conjugated to MACS® MicroBeads and allows for subsequent cell separation.

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

5. Enrichment of specific target cells

In the fifth step the Microbead-labeled IFN-secreting cells of interest are isolated by the built-in magnetic column of the CliniMACS Prodigy. While unlabeled cells pass through, Microbead-labeled cells are being retained in the magnetic field. Afterwards these cells are collected in the target cell bag, ready for analysis and downstream applications. 

Express Modes of the MACSQuant® Instruments allow for fully automated and standardized flow cytometry processes.

6. Quality control

The last step is the analysis of the final cell product. Immune cell composition and target cell numbers are analyzed as well as phenotype and frequency of the target cells. In addition isolated IFN-y positive isolated cells are tested for activity (see expansion and restimulation special protocol, research use only). 

When using a MACSQuant® Flow Cytometer the Virus-Specific T Cell CCS Express Mode Package allows for fully automated and standardized cell analyses. The package contains three optimized application-specific Express Modes and detailed Special Protocols (see related documents) for sample preparation and the use of the Express Modes.

Antibody panels for the Virus-Specific T Cell Express Mode Package:

Express ModePurposeV1 VioBlue®V2 VioGreen™B1 FITCB2 PEB3 7AAD PerCP-Vio® 700B4 PE-Vio 770R1 APCR2 APC-Vio 770
Blood donor eligibility test 
(RCI_CD4CD8)
Determination of IFN-γ  
expression on target cells
CD3-CD8CytCD14/CD20-CD4-
Immune cell composition 
(CCS_Immune_Cell_Composition_h_01, Panel A)
Determination of cellular composition 
and target cell number
CD45CD4CD3CD16/CD567-AADCD19CD14CD8
Purity 
(CCS_Purity_h_01, Panel B)
Determination of phenotype
and frequency of target cells 
CD45CD4CD3IFN-γ 7-AADCD45ROCD62LCD8

References

  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, S. 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: 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, A. 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 afterallogeneic stem cell transplantation. J. Clin. Oncol. 31: 39–48.
  9. Bunos, M. et al. (2015) Automated isolation of primary antigen-specific T cells from donor lymphocyte concentrates: results of a feasibility exercise. Vox Sang. 109: 387–93
  10. Priesner, C. 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, P. 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, N. et al. (2016) Robust production of cytomegalovirus pp65-specific T cells using a fully automated IFN-γ Cytokine Capture System. Blood 128: 5739.
  13. Qian, C. et al. (2017) Curative or pre-emptive adenovirus-specific T cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: a successful phase I/II multicenter clinical trial. J. Hematol. Oncol. 10: 102.
  14. Kállay, K. et. al. (2018) Early experience with CliniMACS Prodigy CCS (IFN-gamma) System in selection of virus-specific T cells from third-party donors for pediatric patients with severe viral infections after hematopoietic stem cell transplantation. J. Immunother. 41: 158–163.
  15. Pello, O. M. et. al. (2017) BKV-specific T-cells in the treatment of severe refractory haemorrhagic cystitis after HLA-haploidentical haematopoietic cell transplantation. Eur. J. Haematol. 98: 632–634.

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