Optimization of cytomegalovirus specific T cell response via flow cytometry

Human Herpesvirus 5 (HHV-5) which is defined as the human Cytomegalovirus (CMV) is located in Herpesviridae family of the Betaherpesvirinae genus of the Cytomegalovirus species. After the primary infection, CMV causes latent infections in endothelial cells, leukocytes and various tissues. In organ transplantation, if donor is CMV seropositive (D +) and the recipient is seronegative (R-), the risk of the primary CMV infection is high in transplant patients and the risk of CMV reactivation and re-infection is high in CMV-seropositive recipients. Latent virus infections are the most important factors that adversely affect the success which is provided by immunosuppressive therapy in transplant. For this reason, timely detection of CMV infection disease which causes the loss of the transplanted organ rejection and even patients is extremely important and required. The cell culture, histopathological examination, serological tests, molecular methods, CMV antigenemia test and immunological tests are used in a wide range in the diagnosis of CMV infection. Although cell culture that is considered the gold standard in the diagnosis of CMV gave place to more rapid and less labor-intensive tests. Thus in the present day, detection of viral load and quantity with molecular methods has taken place in determination of CMV infection in solid organ transplant patients, the selection of patients to be applied in determining the preemptive treatment and follow-up of the treatment. Especially in immunosuppresive patients and latent virus infections, the presence of host response or absence of host response play a key role in determining the clinical condition. The cellular immune response has especially a great function in the prevention of viral infections, and memory CD4+ and CD8+ T lymphocytes are the functional signatures of cellular immunity. In organ transplant patients, the properties of the agent as well as the characteristic of host immune response will provide early detection of infectious disease, monitoring of immunosuppressive therapy and the treatment of the infection. Cellular immunity tests are studied by ELISPOT, Quantiferon and flow cytometry methods. It ıs possible to evaluate the cells according to size and granularity as well as measure functions by cytokine flow cytometry method which is used in our study. One of the strengths of the Mediterranean University Hospital is the success in organ transplantation. This success also gives a responsibility to the laboratory sciences to follow universal-scientific approach in the diagnosis and follow-up of infectious diseases in organ transplant patients. With a sense of responsibility, we have begun viral-specific cellular response research in organ transplant patients in our laboratory. In our study, we have planned the optimization and validation of CMV-specific T-cell response in renal transplant patients with cytokine flow cytometry method and then planned the monitoring of cellular immune responses of clinical cases. To this end, totally for four times we have taken samples before kidney transplantation (0. month), and post-transplant 1st , 3rd and 6th months from twenty-one patients between the ages of 18 and 66 years whom were planned by Mediterranean University Organ Transplantation Center for live-donor kidney transplantation between dates December 2012 - August 2013. During prospective research process all planned samples were collected from twelve patients but we were unable to collect post-transplant 6. months samples from nine patients since. As a control group, healthy CMV-seropositive kidney donors (24 to 66 years, 10 males, 10 females) were used. After completing the optimization and validation process we have studied the tests of patient and control groups. The main stages of our procedure are, isolation of peripheral blood mononuclear cells from whole blood (PBMC), freezing and storing the samples, thawing samples, ex vivo stimulation of lymphocytes with CMV peptides and counting by flow cytometry after functional seperation as a result of CMV-specific IFN-?-producing T cell surface antigens and intracellular cytokine staining staining. Main findings obtained from the flow cytometry is IFN-?-producing CMV-specific CD4+, CD8+ and total (CD4+ + CD8+) T-lymphocyte percentages. These values and the patient's peripheral blood lymphocyte numbers were proportioned and so CMV-specific IFN-?-producing T-lymphocyte number was calculated in peripheral blood and evaluations were made via these numbers. According to obtained findings after completing all tests for patient and control groups, we showed that post-transplant 3rd month peripheral blood lymphocyte values were significantly lower than post-transplant 1st month peripheral blood lymphocyte values. CMV-specific CD4+ lymphocytes were significantly decreased (p=0,003) in post-transplant 3rd month compared to post-transplant 1st month, and no significant difference was observed between other groups. Third month CMV-specific CD4++CD8+ T lymphocytes were significantly decreased (p=0,039) compared to 1st month value. Patients? CMV IgG values before transplantation, age, gender, immunosuppressive and induction therapies administered to patients did not have any significant effect on IFN-? secreting CMV-specific T-lymphocytes. We observed that the one case whom had low level of viremia in the 6th month sample had no cellular immune response to CMV and had high level of CMV-specific IFN-? secretion in six months. In conclusion, with this study, the first time in our hospital and Turkey, we quantitated CMV specific T cells in kidney transplant patients through cytokine Flow cytometry. Our optimisation and validation experiments showed that freezing and storing samples up to six months does not have any quantitative effect on the virus specific T cells. The results of this study will lead to more comphrensive studies in not only solid organ but also in bone marrow transplantation patients