Monitoring of Cytomegalovirus-Specific CD4+ and CD8+ T Cell Responses by Cytokine Flow Cytometry in Renal Transplant Recipients

In spite of the improvements in the clinical management of solid organ transplant (SOT) recipients provided by immunosuppresion and universal prophylaxis, human cytomegalovirus (CMV) infections continue to be one of the most leading causes of morbidity and mortality. Cell-mediated immunity specific to CMV (CMV-CMI) plays an important role in the control of CMV replication. Therefore, monitoring of CMV-specific T-cell response can be used to predict individuals at increased risk of CMV disease. The aim of this study was to investigate the levels of CMV-specific interferon (IFN)-gamma producing CD4(+) and CD8(+) T cells in kidney transplant recipients before and after the transplantation, by cytokine flow cytometry. A total of 21 kidney transplant recipients (14 male, 7 female; age range: 18-66 years, mean age: 34.5 +/- 9.9) who were all CMV seropositive have been evaluated in the study. Blood samples from the patients were obtained before and at the 1(st), 3(rd) and 6(th) months after transplantation. CMV seropositive healthy kidney donors (n = 20) constituted the control group. The main stages of our procedure were as follows; isolation of peripheral blood mononuclear cells from whole blood, freezing and storing of the samples, later on thawing the samples, ex vivo stimulation of lymphocytes with pooled CMV peptides and counting CMV-specific IFN-gamma producing CD4(+) and CD8(+) T cells by flow cytometry following surface and intracellular cytokine staining. Monitoring of the viral load (CMV-DNA) was performed in 10 days intervals in the first 3 months followed by 3 week intervals until 6 months using COBAS AmpliPrep/COBAS TaqMan CMV test system (Roche Diagnostics, USA). The frequencies of pretransplant CMV-specific IFN-gamma producing CD8(+)T cells in patient (3.53 +/- 4.35/mu l) and control (4.52 +/- 5.17/mu l) groups were not statistically different (p= 0.266). The difference between the number of virus-specific CD4(+)T cells in patients (8.84 +/- 9.56/mu l) and those in the control group (8.23 +/- 11.98/mu l) was at the borderline of significance (p= 0.057). The age and gender of the patients and type of antiviral prophylaxis protocols [valgancyclovir (n= 4); valacyclovir (n= 17)] did not have any significant effect on CMV-CMI (p> 0.05). Similarly, induction therapy administered to four patients did not show any effect on CMV-CMI (p> 0.05). CMV-specific immune responses of patients who received different immunosuppression protocols [tacrolimus + mycophenolate mofetil (MMF) + steroid (n= 17); cyclosporine + MMF + steroid (n= 2); mTOR inhibitor + MMF + steroid (n= 2)] were not different (p> 0.05). The number of CMV-specific CD4(+)T cells in all patients were significantly decreased in the 3rd month compared to the 1st month after the transplantation (p=0.003), indicating a relationship with the period of immunosuppressive therapy. In one of the patients who did not have CMV-specific CD4(+) T-cell response but had cytotoxic T-cells (CD8(+) T= 0.6%) before transplantation, CD4(+) T-cell response have developed during monitorization (1.4%, 1.5% and 0.5% in 1st, 3rd and 6th months, respectively), and no viral reactivation was detected. Out of the two patients who had no CD4(+) and CD8(+) T cell response in the 3rd month, one of them developed low level viremia (150 copies/ml) in the 6th month. In this patient the level of CMV-CMI in the 6th month (CD4(+)T + CD8(+)T= 0.9%), have reached higher values than the values obtained before the transplantation (CD4(+)T + CD8(+)T= 0.5%). The viremia was cleared spontaneously in this patient and no antiviral therapy was required. In conclusion, our results suggested that pretransplant and posttransplant monitoring of CMV-specific T-cell responses might be helpful as well as viral load in the clinical management of CMV infection in SOT patients.