Cytomegalovirus-Specific T Cells Isolated by IFN-γ Secretion Assay Do Not Induce Significant Graft-Versus-Host Reactions In Vitro

Transplantation. 2016 Nov;100(11):2352-2361. doi: 10.1097/TP.0000000000001219.


Background: Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage.

Methods: Cytomegalovirus-specific CTLs were isolated by the IFN-γ secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-γ, cytotoxic activity, IFN-γ and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay).

Results: Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-γ and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-γ and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay.

Conclusions: Cytomegalovirus-specific CTLs isolated by the IFN-γ secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.

MeSH terms

  • CD4-Positive T-Lymphocytes / immunology
  • Cytomegalovirus / immunology*
  • Graft vs Host Reaction*
  • Granzymes / metabolism
  • Humans
  • In Vitro Techniques
  • Interferon-gamma / metabolism*
  • T-Lymphocytes, Cytotoxic / immunology*


  • Interferon-gamma
  • Granzymes