Effector memory CD 4 + T-cells and dendritic cells are noninvasive biomarkers of late cellular rejection after kidney transplantation

DOI:10.23873/2074-0506-2017-10-3-207-216 Effector memory CD4 T-cells and dendritic cells are noninvasive biomarkers of late cellular rejection after kidney transplantation A.V. Nosik, S.V. Korotkov, V.V. Smol'nikova, V.Yu. Grinevich, D.Yu. Efimov, M.V. Dmitrieva, A.A. Syantovich, A.A. Dolgolikova, O.V. Kalachik, S.I. Krivenko, I.I. Pikirenya, A.M. Dzyadz'ko, A.E. Scherba, O.O. Rummo Belarusian Medical Academy of Postgraduate Education, 3 Bldg. 3 P. Brovki St., Minsk 220013 Republic of Belarus, Department of Transplantology, 8 Semashko St., Minsk 220045 Republic of Belarus; The Republican Scientific-Practical Center of Organ and Tissue Transplantation at the City Clinical Hospital No. 9, 8 Semashko St., Minsk 220045 Republic of Belarus; City Clinical Pathologoanatomic Bureau, 8 Bldg. 8 Semashko St., Minsk 220045 Republic of Belarus


Introduction
Currently, kidney transplantation (KT) is the most optimal method of renal replacement therapy. Kidney transplant both improves the quality of life of patients with chronic kidney disease, and also significantly prolongs their survival. Kaballo et al. showed that a 5-year mortality risk in patients after kidney transplantation is 47% lower than in patients undergoing renal replacement therapy [1].
One of the most significant factors affecting the results of transplantation is the renal allograft rejection reaction. Despite the immunosuppressive therapy (IT), the incidence of acute rejection in the early post-transplant period is about 10%. A cellular rejection in the renal graft in late postoperative period (over 1 year) occurs even more often, develops in 35% of patients and is classified, according to Nair et al., as a late cellular rejection [2]. The development of the late cellular rejection results in immunological processes in kidney graft becoming chronic, and it also constitutes one of the causes for the graft function loss [3].
Currently, the "gold standard" for diagnosing a graft rejection reaction is a percutaneous needle biopsy. However, this is an invasive procedure and it is associated with the risk of complications which, according to literature reports, occur as frequent as in 5.2% to 12.56%, and can lead to the allograft loss in 0.25% of cases [4]. Neither the laboratory markers traditionally used to diagnose the kidney function, nor the ultrasound examination of the graft have sufficient sensitivity and specificity.
A promising trend in a non-invasive diagnosis of the rejection reaction in KT is a cytofluorometry (CFM). The use of this technique makes it possible to quantify simultaneously the numbers of practically all subpopulations of peripheral blood leukocytes. However, at present phenotypic biomarkers that would reliably reflect immunological processes in the graft have not been identified and no protocols have been developed on the clinical use of CFM as a method for diagnosing the rejection reaction in a renal allograft.
In this regard, the goal of our work was to study the subpopulations of peripheral blood lymphocytes in patients after KT and to develop the CFM criteria for diagnosing a late cellular rejection.

Material and methods
An observational, retrospective, single-center, analytical, comparative study in two groups was conducted in the "case-control" design, and Protocol for Patients after KT, and did not differ between the study participants of the two groups (see Table 1).  The statistical analysis of the quantitative data in groups was made using the Mann-Whitney's U test. The results of the quantitative data comparison are presented as REJ Median (25% quartile to 75% quartile) vs.
STA Median (25% quartile to 75% quartile), the significance level of differences. To compare the qualitative data in the study groups, the χ 2 test was used. The results of the qualitative data comparison are presented as REJ Absolute rate (Percentage) vs. STA Absolute rate (Percentage), the significance level of differences. To identify the correlation between the data types, the Spearman criterion was used for quantitative data, and the association criterion for the qualitative data. The correlation between the qualitative and quantitative data was estimated using a rank-biserial test. The statistical significant difference between the obtained results was set at p ≤ 0.05.

Characteristics of study participants
Among the study participants, an acute graft dysfunction in the long-
The analysis revealed no trends in the difference in the count of the remaining T-lymphocyte subpopulations, as well as in the counts of Blymphocyte and natural killer subpopulations (Table 2).  Thus, Table 2 shows that among the studied lymphocyte subpopulations, the counts of CD4 + effector memory T-cells, as well as myeloid and plasmacytoid dendritic cells, were statistically significantly different between the study groups.

Discussion
The study results have demonstrated that increased counts of CD4 + effector memory T-cells and a decreased number/count of mDc in the blood of kidney transplant recipients are reliable noninvasive markers of late cellular rejection.
We believe that the data we have obtained are not casual and can be explained by the role of T lymphocytes and mDc in the immune response to alloantigens.
So, CD4 + effector memory T-cells serve as some of the main components of an active immunological memory that support the activation and proliferation of donor-specific CD8 + T-lymphocytes and B cells that, in turn, represent the main substrate of the graft rejection [5]. The increase in the count of this T-lymphocyte subpopulation in blood is most likely associated with the increased immune response to the graft alloantigens resulting in the development of the graft rejection and dysfunction.
Dendritic cells are also one of the main initiators of the immune response, ensuring the capture, processing, and presentation of the antigen to T-lymphocytes. In the system of transplantation immunity, the interaction of CD4 + T-lymphocytes with mDc is the main triggering mechanism of the Tcell response. The decrease in the number of mDc in peripheral blood is associated with their migration to the inflammatory focus, i.e. to the graft [6]. It was demonstrated that the detection of mDc in the renal graft tissues directly correlates with the allograft rejection Grade according to Banff classification. At the same time, the mDc density in renal graft tissues in the period of acute rejection is an independent predictor of the graft functional loss within 1 year [6]. Dendritic cells are a resident subpopulation, and apparently because of that, their count lacks increasing in blood of the recipients with an active immune response to alloantigens [8].
From the foregoing it follows that the differences we have found postoperatively in the immunophenotype of peripheral blood lymphocytes between the patients with a normal graft function and those with the developed late cellular rejection are objective and pathogenetically wellgrounded.

Conclusion
The relevance of results we have obtained lies in the possible effective, rapid, and non-invasive diagnosis of the cellular rejection reaction in the renal graft in patients after kidney transplantation in the long-term.
Cytofluorimetry with the assessment of T-lymphocyte and dendritic cell subpopulations can be used for the differentiated diagnosis of renal allograft dysfunction without the risk of causing complications for both the graft and the patient.

Conflict of interests.
Authors declare no conflict of interest.
Financing. The study was carried out without external funding.