Short-term in-vitro expansion improves monitoring and allows affordable generation of virus-specific T-cells against several viruses for a broad clinical application

Abstract

Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8(+) and CD4(+) HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.

Figure 1. Frequency of responses to novel and known HAdV peptides by ELIspot.

Frequency of HAdV-specific T-cells in donors before and after short-term in vitro expansion analyzed by multimers and IFNg-CSA. A) Schematic drawing of the IL-2-based in vitro expansion protocol for ELIspot analysis. Percentage of donors responding to novel B) and known C) A*02-based peptides by ELIspot assay. Black bars represent donors positive, gray bars those negative for the respective allele. Significant differences are indicated (*, p>0.05, Fisheŕs exact test). D) Schematic drawing of the IL-15-based in vitro expansion protocol for FACS analysis. E) Percentages of A*02 ADV-streptamer⁺ T-cells including representative dot plots are given. F) Specific lysis is shown of autologous and allogeneic A*02 mismatched PHA-blasts (target cells), unloaded- or HAdV-A*02 (LLD) peptide-loaded, induced by A*02-multimer-sorted HAdV-T-cells. The total number of “dying” target cells/well and percentage values were evaluated from each sample. Two representative dot plots are shown. G) A*01, A*24, B*07, and B*35 HLA- dependent ADV-specific multimer⁺ (streptamer or pentamer) T-cells among CD8⁺ T-cells, before (day0) and after 6 and 12 days of expansion are shown. Notably, for some donors, multimer analyses were only performed at day 0, 6 and/or 12. H) Subgroup C-derived streptamer staining of PBMCs stimulated with specific peptides for 12 days, representing different adenoviral subgroups, as indicated. The percentage of streptamer⁺ T cells among CD8⁺ is shown. Asterisks represent subgroup recognition.

Figure 2. Analysis of HAdV-specific T-cells in patients during and after allogeneic SCT.

The presence of 6 day expanded or magnetically isolated HAdV-specific T-cells was assessed by A) the percentage of multimer⁺ T-cells among CD8⁺ and by B) events of IFN-γ secreting CD4⁺ and CD8⁺ T-cells during HAdV load, or after ADV clearance in 10 patients. Of note, for most patients, either multimer or IFN-γ assays before or after viral load were performed. Each dot refers to the appropriate patient number, as indicated.C) Detailed analysis of ADV-multimer⁺ T-cells of patient No. 3 and the representative HSC-donor are shown. Dot plots show the percentage of HAdV-multimer⁺ T-cells among CD8⁺ at several time points after clearance of HAdV plasma load. For the last two stainings, cells were measured directly ex vivo and after a 12 instead of 6 day expansion period. The graphs show percentage of HAdV-multimer⁺ T-cells among CD8⁺ (bold line, rectangle), HAdV copies per ml serum (dotted line, diamond) and number of CD3⁺ T-cells/µl blood (bold line, diamond). D) A summarizing diagram + SEM (of patients No. 2, 4, 7, 9 and 10) including representative dot plots of magnetically isolated HAdV-streptamer⁺ T-cells and percentages of their 4 subsets of naïve (N), central (TCM), effector memory (TEM) and effector memory CD45RA⁺ (TEMRA) T-cells are shown.

Figure 3. Generation and detailed phenotypic analysis of seHAdV-T-cells.

PBMCs (5×10⁶/12 well) were expanded for 12 days using the HAdV peptide pool and IL-15, as described in material and methods. A) The total number of PBMCs after expansion, and B) the absolute number of HAdV-multimer-specific T-cells at days 0 and 12, are shown from 24 donors, including mean +SEM and ranges. C) PBMCs (2.5×10⁶/24 well) were CFSE-labeled without stimulation (unst.), or stimulated with the HAdV-peptide pool in the presence of IL-15, as described in Materials and Methods. A summarizing diagram + SEM of 3 donors shows cell number (x1000) (bars) and percentage (above bars) of viable proliferating cells after expansion including a representative histogram.

Figure 4. Phenotypic analysis of seHAdV-T-cells.

A) Percentage values of different cell populations (as indicated, including cytokine-induced killer cells (CIK)) within PBMCs, before (day 0) and after expansion (day12) of 8 donors. Percentage values of TCM (upper left), Naïve (upper right), TEM (lower left) and TEMRA (lower right) T cell populations within CD8⁺ (B), CD4⁺ C), and HAdV-streptamer⁺ T-cells D) on days 0 (white bars) and 12 (black bars). The graph shows mean+SEM of 8 (for CD4⁺ and CD8⁺ T-cells) and 3 (for HAdV-streptamer⁺ T-cells) donors. Notably, for phenotypic analysis of HAdV-streptamer⁺ T cells on day 0 (white bars), beads-based magnetic isolation was performed. Representative dot plots are shown.

Figure 5. Alloreactive potential of PBMCs versus expanded control- and seHAdV-specific T-cells.

CFSE-labeled autologous or allogeneic responder cells (PBMCs or seHAdV-T-cells) were mixed 1∶1 with irradiated stimulator cells (PBMCs) and incubated for 7 to 8 days. A) The graph shows the total number of proliferated viable cells per 96 well plate of 14 different donor/recipient combinations A–N). Proliferation of seHAdV-T-cells or PBMCs in response to autologous (auto) or allogeneic (allo) irradiated PBMCs are shown. In addition, representative examples from donors A and J are given. B) A summarizing graph show mean + SEM from all combinations (except for L,M and N); The percentage of viable proliferating seHAdV-T-cells was set to 100% and calculated based on total cell number. C) seHAdV-T-cells or seMAGE-1A-T-cells (control) and PBMC were mixed with allogeneic irradiated stimulator PBMCs. A summarizing graph shows mean + SEM of 4 combinations. First, total cell number of viable proliferating (CFSE-low) seHAdV-, seMAGE-1A-T-cells and PBMCs/well are calculated. Based on these total cell number, the percentage values were analyzed and compared to allogeneic PBMCs, which was set to 100%. A representative histogram of one donor is shown, including percentage values of proliferated cells. Significance vs HAdV-T-cell line: o, p< = 0.07; *, p< = 0.01.***, p< = 0.001.

Figure 6. Functional and cytolytic activity of seHAdV-T-cells.

seHAdV-T-cells were used for the cytotoxic assay. The percentage values of IFN-γ, CD137 and CD107a expressing seHAdV-T-cells among CD4⁺ and/or CD8⁺ are shown A–D) as indicated. E) The percentage values of IFN-γ and TNF-α among CD3+ cells are shown, including representative dot plots indicating simultaneous expression. F) Representative dot plots of IFN-γ and TNF-α-expressing CD8⁺ T-cells including streptamer⁺ T cells and CD4⁺ T-cells. G) Specific lysis is shown of autologous and allogeneic PHA-blasts (target cells) matched in only a single MHC I, or in only a single MHC II, unloaded (white bar), HAdV-peptide- (grey bar) or HAdV-peptide pool (black bar)-loaded, induced by seHAdV-T-cells. The total number of “dying” target cells/well was evaluated from each sample. Based on these, a summarizing graph shows the percentage of dying target cells related to unloaded autologous (auto) target cells which is set to 100%. Representative dot plots, indicating “dying” cells/well, are shown. H) Specific lysis of allogeneic, unloaded (white bar) or HAdV-peptide pool (black bar)-loaded target cells induced by post-thaw seHAdV-T-cells. Based on total cell number of dying cells, a summarizing graph shows the percentage of dying target cells related to unloaded target cells, which is set to 100%. Significance vs unloaded autologous targets: ns = not significant, *, p< = 0.01.***, p< = 0.001.