Fig 1.
CMV-specific T cells with TCM phenotype are rarely detectable in peripheral blood.
Four representative adoptive CMV-ACT donors are depicted. (A) CMV epitope-specific CD8+ T cells were quantified by staining with CMV pp65-based HLA-A*02:01/NLV- (donor 1), HLA-A*01:01/YSE- (donor 2), HLA-B*07:02/TPR (donor 3) and CMV IE-1-based HLA-A*02:01/VLE (donor 4) MHC-multimers, respectively. (B) Phenotypic characterization of corresponding MHC-multimer-positive populations (black) was performed according to expression of CCR7 and CD45RO. Phenotypes were defined as TN (naive T cells, CCR7+), TCM (central memory T cells, CD45RO+/CCR7+), TEM (effector memory T cells, CD45RO+/CCR7-) and TEMRA (effector T cells, CD45RO-/CCR7-). Phenotypic distribution of the corresponding CD3+ T cell populations (grey) is shown.
Fig 2.
Peptide-specific proliferation assay (PSPA) for detailed analysis of antigen-specific TCM.
(I) TCM are co-cultured with gamma-irradiated peptide-labeled autologous PBMC (1:6 ratio) in a flat-bottom well plate on day 0. (II) Cells can be restimulated with corresponding stimulator PBMC on day 7. IL-2 (50 IU/ml) is added at day 3 and thereafter every 3–5 days IL-2-containing medium is used for culture medium exchange. (III) Cells can be primarily harvested on day 12–21. After harvest, they either undergo stimulation with single peptides or peptide mixes followed by ICS or are stained directly by MHC-multimers.
Fig 3.
Minimal numbers of virus epitope-specific TCM undergo extensive proliferation and phenotype differentiation.
Human PBMC-derived CD3+ T cells of three healthy donors (Donor 1–3) were enriched using magnetic CD3-Fab-Streptamers. TCM were subsequently isolated by FACS-sorting using anti-CD45RO and -CD62L fluorescent antibodies. Finally, isolated TCM underwent a PSPA using CMV pp65-based HLA-A*02:01/NLV peptide. Ex-vivo-staining of PBMCs before enrichment and sorting is shown (before TCM sort). CD3+ (grey) and CMV MHC-multimer+ CD8+ T cells (black) were analyzed with regard to their TN (CD45RO-/CD62L+), TCM (CD45RO+/CD62L+), TEM (CD45RO+/CD62L-) and TEMRA (CD45RO-/CD62L-) phenotype (far left column). Relative size of MHC-multimer+ TCM is depicted (middle left column). After FACS-sorting and long-time CMV peptide stimulation (after TCM sort and PSPA), cultured TCM were analyzed regarding MHC-multimer+ CD8+ T cell frequency (middle right column) and phenotype differentiation (far right column).
Fig 4.
PSPA of GMP-grade TCM products enables visualization of multiple, ex vivo undetectable T cell populations against various viruses.
One representative non-mobilized leukapheresis product from a healthy donor is depicted. After performed leukapheresis, TCM were generated the following day in a GMP-compatible process via two-step positive enrichment using CD3-Fab-Streptamers and CD62L-Fab-Streptamers, respectively, followed by a depletion with CD45RA-Fab-Streptamers. Subsequently, isolated TCM underwent a PSPA using AdV Hexon-based and CMV pp65- and IE-1-based single peptides with restriction to HLA-B*07:02, -C*07:02 and A*24:02, as well as EBV EBNA3c peptide pool. Ex-vivo-staining of original donor PBMCs is shown (before TCM isolation). CD3+ (grey) and virus MHC-multimer+ CD8+ T cells (black) or IFNγ+ CD8+ T cells (black, bottom row) were analyzed with regard to their TN, TCM, TEM and TEMRA phenotype (far left column). Relative sizes of MHC-multimer+ CD8+ TCM or IFNγ+ CD8+ TCM (bottom row) are depicted (middle left column). After TCM isolation and long-time peptide stimulation (after TCM isolation and PSPA), cultured TCM were analyzed regarding MHC-multimer+ and IFNγ+ (bottom row) CD8+ T cell populations (middle right column) and phenotypical differentiation (far right column).
Fig 5.
Virus epitope-specific TCM can be generated from mobilized stem cell apheresis material.
Highly pure (>95%) human mobilized stem cell apheresis-derived TCM was generated by FACS-sorting using anti-CD3, -CD45RO and–CCR7 fluorescent antibodies. Subsequently isolated TCM underwent a PSPA using AdV hexon-based HLA-A*01:01/TDL and HLA-A*01:01/LTDL peptides as well as AdV hexon 5 peptide pool (A*24:02/TYF population). Ex-vivo-staining of unsorted donor PBMC is shown (before TCM sort). CD3+ (grey) and AdV MHC-multimer+ CD8+ T cells (black) were analyzed with regard to their TN, TCM, TEM and TEMRA phenotype (far left column). Relative size of MHC-multimer+ TCM is depicted (middle left column). After FACS-sorting and long-time AdV-peptide stimulation (after TCM sort and PSPA), cultured TCM were analyzed regarding MHC-multimer+ CD8+ T cell frequencies (middle right column) and phenotypical differentiation (far right column).