ABSTRACT
Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating 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(+) 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)-γ 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(+) CTLs, which was confirmed based on the reduced production of IFN-γ 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, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ 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(+) T cells and affects various elements of T cell activation.
Subject(s)
Antigen-Presenting Cells/drug effects , Granulocyte Colony-Stimulating Factor/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Regulatory/drug effects , Antigen-Presenting Cells/cytology , Antigen-Presenting Cells/immunology , CD28 Antigens/genetics , CD28 Antigens/immunology , CD3 Complex/genetics , CD3 Complex/immunology , Cell Communication/drug effects , Cell Communication/immunology , Cell Line, Tumor , Coculture Techniques , Gene Expression Regulation , Granzymes/antagonists & inhibitors , Granzymes/genetics , Granzymes/immunology , Humans , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/genetics , Interferon-gamma/immunology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/immunology , MicroRNAs/genetics , MicroRNAs/immunology , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/immunology , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/immunology , Primary Cell Culture , RNA, Messenger/genetics , RNA, Messenger/immunology , Signal Transduction , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunologyABSTRACT
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.
