Foxp3 Gene Expression Value in Regulatory T Cells in Pathogenesis of Graft-Versus-Host Disease Induced with Cryopreserved Allogenic Material

Content of regulatory T cells (Treg) and their Foxp3 gene expression level in the recipients with graft-versus-host disease (GVHD) induced by introduction of fresh aspirated or cryopreserved allogenic bone marrow combined with lymph node cells were under study in this research. In GVHD experimental model the deficiency in Tregwas shown to play an important role in maintaining autoimmune process. In the animals with GVHD induced with fresh aspirated allogenic bone marrow (BM) almost twofold decrease in both Tregcontent and the expression level of Foxp3 gene were established as compared to the syngeneic BM recipients. An increased level of Foxp3 gene expression under the maximum reduction of their content in the animals with GVHD, induced with cryopreserved allogenic BM introduction, was noted. Cryopreservation of allogenic BM, used for GVHD induction, reduced its immune reactivity and, as a result, clinical evidence of pathology. Our findings enable to broaden the notion about the mechanisms of immune conflicts development in GVHD both at cellular and molecular levels.


Материалы и методы
Исследования проводили на мышах линий СВА/Н и (СВА/Н×С57Вl) F1 20-недельного возраста массой 24-26 г в соответствии с «Общими принципами экспериментов на животных», одоб-Studying the mechanisms of graft-versus-host disease (GVHD) progress and prevention is stipulated by the extending of application scope for hematopoietic tissue to treat radiation sickness, malignancy and immunodeficiency disorders. In all the cases the main obstacle for successful engraftment of transplanted tissue is GVHD, in pathogenesis of which a key role is played by regulatory T cells (T reg ) [8,17,19]. Their main function was established to consist in suppressing the activity of autospecific clones of T-lymphocyte, thereby preventing the autoimmune processes. Therefore, the disorder in T reg structure and function is commonly assumed to play an important role in pathogenesis of various diseases [8,15]. Prospects of using these cells as an adaptive therapy in autoimmune diseases are currently under consideration [13]. In experimental murine models the T reg deficiency, not being the main factor of pathogenesis, was demonstrated as playing an important role in autoimmune process maintenance [15]. One associates Foxp3 gene expression with the population of regulatory T cells. Miura Yu. et al. [16] demonstrated the Foxp3 expression to be significantly reduced in peripheral blood mononuclear cells of patients during GVHD progress, wherein the level of its expression was inversely proportional to the pathology severity. FOXP3 is known to be the most specific intracellular marker for T reg , and Foxp3 gene is responsible for their development and a suppressive function. That is exactly why it is necessary to study the immune mechanisms of GVHD development not only at cellular level (T reg content), but at molecular and genetic ones as well (Foxp3 gene expression).
Due to a need in creating bone marrow stocks to treat a radiation sickness and other hemodeficits of a body, its cryopreservation and storage at liquid nitrogen temperature (-196°C) are essential. Therefore, in our studies we used the cryopreserved allogenic BM for introduction to lethally irradiated animals.
This research was aimed to assess T reg content and Foxp3 gene expression rate in the cells of recipients with GVHD induced by an introduction of fresh aspirated or cryopreserved allogenic bone marrow with lymph node cells.

Materials and methods
Research was carried out in CBA/H and (CBA/ H×C57Bl) F1 mice of 20 weeks age weighing 24-26 g, according to the General Principles of Experiments in Animals approved by the 5 th National Congress in Bioethics (Kyiv, 2013) and agreed to the statements of European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasburg, 1986).
Интенсивность развития БТПХ оценивали на 14-е сутки развития патологии по индексу селе-homogenizer with medium 199 (Chumakov Institute of Poliomyelitis and Viral Encephalitides of Russian Academy of Medical Sciences, Russia) supplemented with 3% fetal bovine serum (BioloT, Russia) and 2% sodium citrate (hereafter the handling medium). BM was cryopreserved under protection of 10% dimethyl sulfoxide (DMSO) (Arterium Corporation, Ukraine) in 1.8 ml plastic vials (Nunc, Germany) with 2×10 6 cells/ml concentration using the programmed freezer UOP-6 (Institute for Problems of Cryobiology and Cryomedicine of NAS of Ukraine, Kharkov) by the two-step program: cooling rate of 1 deg/min down to -25°C and following immersion into liquid nitrogen (-196°C) [1]. Stored for a month the BM cells before introduction to the irradiated recipients were thawed in 41°C water bath up to solid phase disappearance. Cells were one-fold washed of DMSO by a slow adding of an equal volume of handling medium and a subsequent 10-min centrifugation (200g). BM cell suspension not subjected to freeze-thawing hereinafter will be referred as the 'native' one.
GVHD was induced as follows. The (CBA/ H×C57Bl)F1 mice were irradiated using RUM-17 device (Mosrentgen, Russia) in 850 R dose. The irradiation conditions were as follows: dose rate of 38.6 R/min; 220 kV voltage; 10 mA current intensity; 0.5 mm Cu + 1 mm Al filters; 50 cm focus-dorsal distance. One hour later irradiation the animals were intravenously injected with 5×10 6 cells/mouse of either native or cryopreserved BM with CBA/H lymph node cells in ratio of 3:1 respectively. The (CBA/H×C57Bl) F1 intact mice before and after administering 5×10 6 cells/mouse of syngeneic BM were used as the control.
The animals were divided into the following groups: group 1 was the syngeneic control (syngeneic BM introduction); group 2 -GVHD (nBM + ln), GVHD induction with administering an allogenic native BM together with lymph node cells; group 3 -GVHD (cBM+ln), GVHD induction with introducing allogenic cryopreserved BM together with lymph node cells; group 4 -the intact control.
The intensity of GVHD development was assessed to day 14 of pathology development by the spleen index (SI), survival rate and regulatory T cells content. Spleen index was calculated as the ratio of organ mass to body one of an animal. The spleen index of intact mice was assumed as 1, the index higher than 1.3 indicated the GVHD development [7,10].
The Foxp3 gene expression level was evaluated in CD4 + fraction of spleen cells in GVHD animals. These cells from the recipient spleens were isolated via im-mune magnetic separation with magnetic sorter (BD Imagnet, USA) using the anti-mouse CD4 magnetic particles: Magnetic Particles -DM (BD Biosciences, USA) by the corresponding protocol. The content of Foxp3 gene transcripts was determined with the rever-se transcription polymerase chain reaction (RT-PCR). To isolate nucleic acids from 1×10 5 CD4 + cells we used Diatom RNA Prep 100 sets (Isogene Lab, Russia), containing the lysing reagent, guanidine thiocyanate. RT-PCR reaction was performed using the set of random-oligonucleotides and reverse transcriptase (M-Mlv) according to instructions of the manufacturer (Reverta L, Russia). The primers of Foxp3 gene (NM_054039.2) and housekeeping gene beta actin (NM_007393.3) were designed basing on the National Center for Biotechnology Information (NCBI BLAST, USA) database and synthesized in CJSC Medbioservis (Ukraine). The amplification of DNA fragments was performed in Tercyc Conventional PCR thermal cycler (DNA-Technology, Russia), the denaturation was done at 94°C for 30 sec, matrix hybridization with a primer was performed at 60°C within 30 sec, and the elongation was done at 72°C for 60 sec. After PCR completing the elongation was done at 72°C for 5 min. Number of cycles was 40. Transcript number of the studied genes was detected using the capillary electrophoresis with chip-analyzer Agilent 2100 (Agilent Technologies, USA), based on the relative semi-quantitative assessment of amplified products [6]. Chips were prepared according to instructions of DNA 1000 kit (Fermentas, Lithuania). The results were normalized in respect to the index of housekeeping gene beta actin expression: internal control for PCR.
Поскольку селезенка -основной орган, в котором формируются зрелые элементы Т-ряда, в нем characteristic feature is an increased SI (Fig. 1). Thus, the SI in syngeneic BM recipients was not significantly different from the intact control, but for the recipients of group 2 (GVHD (nBM + ln)) it was 1.7 times higher. In group 3 animals (GVHD (cBM + ln)) the SI increased to a lesser extent than in group 2, since the immune reactivity of allogenic BM reduced during freezing [2]. However, this index in this animal group increased in 1.5 times as compared to the intact control, that also testified to the GVHD development. Of note is the fact, that both SI value and the survival rate of animals are stipulated by pathology severity (Fig. 2) [1].
Actually the survival rate in recipients was established to correlate with the SI value. Thus, a lower value of SI in group 3 animals than in group 2 correlated with higher index of their survival.
The development of autoimmune processes, also accompanying the GVHD, stipulates a decrease in a recipient body of a number and functional activity of T reg , suppressing the activity of autospecific effector T cells [8]. The state of regulatory T immunity link in recipients with various forms of transplanted material was evaluated to day 14, i.e. when clinical and laboratory indices confirmed the GVHD development (see Figs. 1 and 2).
CD4 + CD25 + FOXP3 + T reg is the 'minor' one; their content in peripheral blood makes approximately 5-10% of CD4 + T cells in mice and humans [8,9]. According to our data the T reg content in spleen of intact mice corresponded to this index. Regulatory T cells play a key role in an immune system due to the unique capability for immune response control. Due to their insufficient number the autoimmune diseases develop and the survival rate in recipients is decreased [4,14].
GVHD is known as associated with the appearance in a body of 'autoreactive' clone of effector T cells (CD4 + ), formed by donor stem hematopoietic cells [5], and a cytokine 'storm', caused by its development, induces the expression of surface markers both on effector and regulatory cells [16]. The expression of CD25 marker under the effect of transforming growth factor beta and interleukin-2 in CD4 + CD25 -T cells transfers them into the T reg category with suppressor activity [19].
У животных группы 2 по сравнению с группой 1 (сингенный контроль) (см. рис. 3), содержание Т рег уменьшалось: CD4 + CD25 + -на 28%, а FOXP3 + -content in FOXP3 + cells was not significantly changed, that might testify to their functional activity, being adequate to the intact control. In group 2 recipients there was noted a decreased content of CD4 + CD25 + and FOXP3 + cells in 1.4 and 1.6 times, respectively, as compared to group 1 (Fig. 3). The MFI index of T reg was also reduced in recipients of group 2, testifying to a change in functional activity of these cells. Our findings were confirmed in the report of Chen X. et al. [12], where they noted a progressive reduction of CD4 + CD25 + FOXP3 + cells content in spleens of recipients with GVHD, caused by administered allogenic BM with splenocytes.
Clinical practice usually involves not autologous (syngeneic) BM, but cryopreserved allogenic one, therefore of interest was to determine the content of T reg and their MFI when inducing GVHD with allogenic cBM+ln (group 3). Thus, the content of the studied T reg subpopulations in this group of animals decreased in a greater extent than when nBM + ln (group 2) was administered, but the indices of their MFI did not significantly differ. This fact indicates the higher functional activity of Treg at their lower amount (Fig. 3).
Of note is the fact, that T reg play a major role in controlling GVHD development: a decrease in this cell population and a disorder in its functional status strengthen clinical symptoms of pathology, in particular, reduce the survival rate in animals. To decode the mechanisms of GVHD development it is important to perform studies both at cellular and molecular levels. So the next step of our research was to evaluate the Foxp3 gene expression level, determining a suppressor function of T reg .
Our findings are consistent with those of Miura Yu. et al. [16], who found out that the mRNA expression of Foxp3 gene was reduced in mononuclear cells of peripheral blood of patients with allogenic and autologous GVHD as compared to the recipients in which no GVHD was developed. The authors demonstrated the Foxp3 gene expression to be inversely related to the severity degree of pathology, also confirming our results (see Fig. 1, 2, and 4).
In animals of group 3 the T reg content was even lower than in group 2, but the expression of the studied gene was higher. An increase in the expression level of Foxp3 gene in group 3 (contrary to group 2) evidently results from the compensation of functional activity of T reg low content. Proceeding from Miura Yu. et al. report [16], the augmentation of Foxp3 gene expression results in a decreased GVHD severity. In our study this fact is confirmed by the survival rate in group 3 animals (see Fig. 2).
There was reported that Foxp3 might be expressed by the effector T cells after activation [11], but the expression of Foxp3 by CD4 + CD25 -T helper cells was not always accompanied by the gaining the suppressor function and a stable regulatory phenotype [18].
Thus, our findings are consistent with the data reported [11,16], demonstrating the correlation between the T reg content and expression level of Foxp3 gene as not always existing. For example, in patients with chronic autoimmune thyroiditis on the background of a constant content of CD4 + CD25 hi cells in peripheral blood the expression level of Foxp3 gene was established to be 1.6 times lower than in healthy patients [3]. It should be noted that only in human the CD4 + CD25 hi cells in CD4 + CD25 + population are true T reg [9]. The authors concluded that an increased number of CD4 + CD25 hi cells under this pathology might be a compensatory response of their functional deficiency, resulting from a reduced Foxp3 expression.