No abstract

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.

SummaryBesides mobilizing stem cells into the periphery, granulocyte colonystimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8 1 T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-g and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8 1 CTLs, which was confirmed based on the reduced production of IFN-g and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocytespecific protein tyrosine kinase (Lck) and CD3f after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8 1 T cells and affects various elements of T cell activation.

Heme oxygenase (HO)-1, the inducible isoform of HO, has immunomodulatory functions and is considered a target for therapeutic interventions. In the present study, we investigated whether modulation of HO-1 might have regulatory effects on in-vitro T cell activation. The study examined whether: (i) HO-1 induction by cobalt-protoporphyrin (CoPP) or inhibition by tin-mesoporphyrin (SnMP) can affect expansion and function of virus-specific T cells, (ii) HO-1 modulation might have a functional effect on other cell populations mediating effects on proliferating T cells [e.g. dendritic cells (DCs), regulatory T cells (T(regs)) and natural killer cells] and (iii) HO-1-modulated anti-viral T cells might be suitable for adoptive immunotherapy. Inhibition of HO-1 via SnMP in cytomegalovirus (CMV)pp65-peptide-pulsed peripheral blood mononuclear cells (PBMCs) led to increased anti-viral T cell activation and the generation of a higher proportion of effector memory T cells (CD45RA(-) CD62L(-)) with increased capability to secrete interferon (IFN)-γ and granzyme B. T(reg) depletion and SnMP exposure increased the number of anti-viral T cells 15-fold. To test the possibility that HO-1 modulation might be clinically applicable in conformity with good manufacturing practice (GMP), SnMP was tested in isolated anti-viral T cells using the cytokine secretion assay. Compared to control, SnMP treatment resulted in higher cell counts and purity without negative impact on quality and effector function [CD107a, IFN-γ and tumour necrosis factor (TNF)-α levels were stable]. These results suggest an important role of HO-1 in the modulation of adaptive immune responses. HO-1 inhibition resulted in markedly more effective generation of functionally active T cells suitable for adoptive T cell therapy.