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 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.
Antibody panels for the Virus-Specific T Cell Express Mode Package:
|Express Mode||Purpose||V1 VioBlue®||V2 VioGreen™||B1 FITC||B2 PE||B3 7AAD PerCP-Vio® 700||B4 PE-Vio 770||R1 APC||R2 APC-Vio 770|
|Blood donor eligibility test |
|Determination of IFN-γ |
expression on target cells
|Immune cell composition |
(CCS_Immune_Cell_Composition_h_01, Panel A)
|Determination of cellular composition |
and target cell number
(CCS_Purity_h_01, Panel B)
|Determination of phenotype|
and frequency of target cells
For the manufacture and use of the antigen-specific T cells, national and international legislation and regulations must be followed.
Miltenyi Biotec as the provider of the CliniMACS CCS-IFN System does not give any recommendation regarding the use of the manufactured cells for therapeutic purposes and does not make any claim regarding a clinical benefit. For complete regulatory notes, please click here.
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