Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein-Barr virus-specific T cells as targets of interest in immunotherapeutic approaches.

Adoptive immunotherapy with donor-derived antiviral T cells can prevent viral complications such as with cytomegalovirus (CMV) and Epstein-Barr virus (EBV). In this context accurate monitoring of cellular immunity is essential and requires suitable quantitative and qualitative assays for high-throughput screening. We comparatively analyzed 57 HLA-typed healthy donors for memory T-cell responses to CMV- and EBV-derived proteins, peptide pools and single HLA-restricted peptides by five commonly used immunoassays in parallel: enzyme-linked immunospot (ELISPOT), cytokine secretion assay (CSA), intracellular cytokine staining (ICS), enzyme-linked immunosorbent assay (ELISA) and pMHC multimer staining. T-cell responses varied greatly between the different target antigens in the investigated assays. IFN-γ ELISPOT consistently detected the highest T-cell response levels against CMV and EBV. CMV-specific T cells were detected in 100% of CMV-seropositive donors tested using CMVpp65 protein and/or overlapping CMVpp65 peptide pool. CMV-specific T cells in HLA-A*02:01-positive/CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 HLA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected. Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells (<92.9% peptides, <56.3% peptide pool) were more frequent than EBNA-specific T cells (<64.3% peptides, <46.9% peptide pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide epitopes was predominant (multimer staining: EBNA3A: 9/14 and BZLF1: 7/14, IFN-γ ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65. Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization of each IFN-γ-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis of antigen-specific T-cell subsets.

Impaired functionality of antiviral T cells in G-CSF mobilized stem cell donors: implications for the selection of CTL donor.

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells. CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry. G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF. To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.