Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment

Abstract

SUMMARYThe availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8 + T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8 + T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferongamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8 + T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0·005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0·005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.

Functional analysis of cytomegalovirus-specific T lymphocytes compared to tetramer assay in patients undergoing hematopoietic stem cell transplantation

“…[6][7][8] Others have shown that the frequency and the absolute number of CMV-specific CD8 þ T cells were similar in patients receiving corticosteroids and those who didn't, while the CMV-specific CD8 þ T cells showed decreased cytokine production. 10,11 Our result was consistent with the latter observation that while the number of CMV-specific CTL does not decrease significantly with corticosteroid therapy, IFN-g production of CMV-specific CTL is severely suppressed. Therefore, concomitant assessment of T-cell function is essential in patients after HSCT, especially in those who are receiving corticosteroid therapy.…”

“…For this purpose, tetramer-based monitoring of CMV-specific cytotoxic T-cells (CTL) has been widely performed in patients with an HLA-A02 or HLA-B07 serotype. [3][4][5][6][7][8][9][10][11] Some of the results have demonstrated that the reconstitution of CMV-specific CTL as evaluated by quantitative tetramer to levels 410-20/ml is adequate for protection against CMV infection. [5][6][7] However, some patients with CMV-specific CTL above this level still experience CMV reactivation.…”

“…[5][6][7] However, some patients with CMV-specific CTL above this level still experience CMV reactivation. 9 It has also been reported that the cellular response to CMV in immunosuppressed patients reflects functional impairment, 10 and CMV reactivation following HSCT has been shown to be associated with the presence of dysfunctional CMV-specific T-cells. 11 Therefore, by itself, the quantification of CMV-specific CTL seems to be insufficient and a simultaneous qualitative analysis of CMVspecific lymphocytes is needed.…”

Characterization of cytomegalovirus pp65-HLA-A24 peptide-specific CTL lines from metastatic melanoma patients

“…Probable reasons for this are as follows: i) melanoma patients are much older than volunteers and have high anti-CMV antibody titers, and ii) CTL killing activity was not impaired in the melanoma patients shown in Fig. 6, in contrast to a report that CMV-specific CTLs in immunosuppressive patients showed a decreased functional response to CMV-peptide (24). Our previous report (25) that autologous DC-mediated stimulated CTLs against HLA-A24 melanoma peptide cocktail showed less potent killing activity than healthy volunteers might demonstrate the greater immunogenicity of CMVspecific CTL epitopes.…”

Monitoring cytomegalovirus IE‐1 and pp65‐specific CD4+ and CD8+ T‐cell responses after allogeneic stem cell transplantation may identify patients at risk for recurrent CMV reactivations

“…Here, the use of corticosteroids impaired these functions in a dose-dependent way. In addition, the combined use of cytokine flow cytometry and class I HLA multimers has revealed that steroid treatment reduces the cytokine-producing fraction within the CMV-specific CD8 1 T cells (42,47). The combined analysis of these studies and our results show that the reduction of CMV-specific T cells in number and function, as an unavoidable effect of immunosuppression to control alloreactivity, will pose SCT recipients at an increased risk of CMV reactivation.…”