CC BY
89
ACTIVATION OF CD4+CD39+ Т CELLS IN COLORECTAL CANCER
Zhulai GA1 Churov AV1, Oleinik EK1, Romanov AA2, Semakova PN1, Oleinik VM1
1 Institute of Biology, Karelian Research Center of the Russian Academy of Sciences (IB KarRC RAS), Petrozavodsk
2 Republic Oncology Center, Petrozavodsk
Pathogenesis of colorectal cancer (CRC) is accompanied by significant changes in the immune system. However, the role of the adenosine-A2AR-mediated immunosuppressive pathway in oncogenesis and more specifically, the expression of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1, also known as CD39) remains unclear. The aim of this work was to study the role of CD4+ T cells, most importantly CD39-expressing regulatory T cells (Tregs) in the formation of immune suppression in CRC and in patients with acute pancreatitis (AP). Expression of CD39 by peripheral blood lymphocytes and tumor-infiltrating lymphocytes (TILs) was measured by flow cytometry. The levels of CD39 messenger RNA (mRNA) in the peripheral blood leukocytes were determined by real-time PCR. Our study reveals that patients with CRC accumulate peripheral CD4+CD39+ cells in the advanced stages of the disease. The proportion of CD39-expressing CD4+ T cells in the total pool of TILs was higher than in the peripheral blood of the same patients. Tregs of both peripheral blood and tumor specimens of CRC patients showed increased CD39 expression. We have found reliable correlations between the levels of CD4+CD39+ T cells and the parameters of cell-mediated immunity in CRC patients. Also, CD39 mRNA levels gradually increased during CRC progression. In contrast, patients with AP have the same levels of CD39 mRNA and peripheral blood CD4+CD39+ T cells as the controls. Finally, we conclude that activation of CD4+CD39+ T cells has an important role in oncogenesis and needs to be studied further.
Keywords: colorectal cancer, ectonucleotidase CD39, Treg cells, transcription factor FOXP3
Funding: the study was part of the State assignment for the Federal Research Center Karelian Research Center of the Russian Academy of Sciences (Project 0221-2017-0043).
1X1 Correspondence should be addressed: Galina A. Zhulai
Pushkinskaya 11, Petrozavodsk, Republic of Karelia, 185014; [email protected]
Received: 25.01.18 Accepted: 20.03.18
DOI: 10.24075/brsmu.2018.027
АКТИВАЦИЯ CD4+CD39+ Т-КЛЕТОК ПРИ КОЛОРЕКТАЛЬНОМ РАКЕ
Г. А. Жулай1ЕЗ, А. В. Чуров1, Е. К. Олейник1, А. А. Романов2, П. Н. Семакова1, В. М. Олейник1
1 Институт биологии, Карельский научный центр Российской академии наук (ИБ КарНЦ РАН), Петрозаводск
2 Республиканский онкологический диспансер, Петрозаводск
Патогенез колоректального рака (КРР) сопровождается значительными изменениями в состоянии иммунной системы. Однако роль аденозин-А2АЯ-опосредованного иммуносупрессорного механизма и в частности экспрессии молекулы эктонуклеозидтрифосфатдифосфогидролазы-1 (ЕЫТР01), или СЭ39, в его развитии до конца не изучена. Целью работы было исследование роли СЭ4+ Т-клеток, прежде всего экспрессирующих СЭ39 регуляторных Т-лимфоцитов (Тгед), в формировании иммунной супрессии при КРР, а также у больных острым панкреатитом (ОП). Экспрессию молекул лимфоцитами крови и опухоль-инфильтрирующими лимфоцитами (ОИЛ) анализировали методом проточной цитометрии. Содержание матричной РНК (мРНК) CD39 в лейкоцитах периферической крови определяли методом полимеразной цепной реакции (ПЦР) в реальном времени. В результате исследования показано, что у больных КРР накопление периферических С04+С039+ клеток происходит на поздних стадиях развития опухоли. Среди ОИЛ количество СЭ4+ Т-клеток, экспрессирующих молекулу СЭ39, выше, чем в крови тех же больных. Значительно повышен уровень экспрессии этой молекулы у регуляторных Т-клеток (Тгед) больных КРР как на периферии, так и среди ОИЛ. Установлены достоверные связи между содержанием С04+С039+ Т-клеток и показателями клеточного иммунитета больных КРР. Обнаружено, что уровень мРНК гена CD39 также увеличивался в процессе развития КРР. У больных ОП, напротив, содержание мРНК гена CD39 оставалось на уровне контроля, так же как и количество С04+С039+ Т-клеток в периферической крови. Таким образом, можно заключить, что активация С04+С039+ Т-клеток в процессе канцерогенеза играет важную роль и требует дальнейшего изучения.
Ключевые слова: колоректальный рак, эктонуклеотидаза СЭ39, Тгед-клетки, транскрипционный фактор РОХР3
Финансирование: исследование выполнено в рамках государственного задания КарНЦ РАН, тема № 0221-2017-0043.
ЕХ] Для корреспонденции: Галина Анатольевна Жулай
ул. Пушкинская, д. 11, г Петрозаводск, 185014; [email protected]
Статья получена: 25.01.18 Статья принята к печати: 20.03.18
DOI: 10.2 40757vrgmu.2018.027
Colorectal cancer (CRC) is one of the most common malignancies and causes of death in Russia [1] and across the world [2, 3]. The number of patients with primary CRC is constantly growing; interestingly, its incidence is much higher in industrial Europe and North America than in the developing countries of Africa, Asia and Latin America [2]. CRC formation is closely associated with the mechanisms regulating the
immune response and is accompanied by the infiltration of immunocompetent cells into the tumor [3-5]. The role of chronic inflammation in promoting CRC is being actively discussed at the moment, as patients with inflammatory intestinal conditions turn to be at a higher risk of developing CRC. According to some reports, anti-inflammatory therapies reduce the risk of gastrointestinal cancer [7, 8].
At present, cancer immunology research is pretty much focused on ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1, CD39). Together, CD39 and CD73 (ecto-5'-nucleotidase, NT5E) participate in the production of extracellular adenosine. Synthesis of extracellular purine nucleosides plays a crucial role in the regulation of inflammation and tissue homeostasis. Immunocompetent cells receive the adenosine signal through A2AR, one of four adenosine G-protein-coupled receptors. Stimulation of A2AR in lymphocytes causes a decline in IL-2 secretion and proliferative activity of native CD4+ T cells, leads to a cutdown in the production of IFN-y and IL-4 by T helper cells and results in the increased expression of CTLA-4, PD1 and CD-40L molecules [8]. This mechanism of immune suppression, involving adenosine-A2AR interactions, can protect healthy tissues against damage induced by inflammation. However, the adenosine-A2AR signaling pathway is also activated in malignant tissues, especially in response to hypoxia, allowing cancer cells to evade recognition by the immune system and, therefore, escape elimination [9]. The role of this mechanism in oncogenesis has been demonstrated in A2AR-deficient mice that rejected immunogenic tumors [10] and also in mice with knocked-out CD39 and CD73 that acquired different cancer-resistant phenotypes [11, 12].
Expression of CD73 in malignant tissues is well described. CD73 is known to be expressed by endothelial, stromal and tumor cells [13]. Less is known, however, about the expression of the membrane marker CD39 in the microenvironment of the tumor. Presumably, regulatory T cells (Tregs) are one of the major sources of CD39 in tumor infiltrates [14]. Using a variety of suppressive mechanisms, Tregs can block the autoimmune response and sustain immune tolerance [15, 16]. The master transcription factor for these cells responsible for their growth and suppressive function is FOXP3 [17]. The role of Tregs in cancer formation is detrimental since they contribute to the disease progression. Tregs have been shown to accumulate in the peripheral blood of cancer patients and tumor tissues [18].
At present, CRC remains one of the most common type of malignancies, but the role of the immunosuppressive adenosine-A2AR pathway in its formation is still understudied. The aim of our work was to explore the role of CD39-expressing CD4+ T cells in the development of immune suppression in patients with CRC.
METHODS
Our study was conducted in 42 patients aged between 18 and 70 years (mean age of 65 ± 12.4 years) with a histologically and cytologically confirmed diagnosis of colorectal cancer. Patients with a previous history of other cancers or immunoinflammatory disorders were excluded from the study. Lymphocytes of 30 healthy donors aged 54.4 ± 20.6 years were used as a control. The CRC diagnosis was established based on clinical, laboratory, endoscopic and morphological tests. Six patients were diagnosed with stage I CRC (14.3%); 15, with stage II (37%); 12, with stage III (28.6%), and 9 patients had stage IV (20%). The patients were divided into two groups: the first group included patients with stages I and II CRC; the second consisted of patients with stages III and IV of the disease. The study was approved by the Ethics Committee affiliated with the Ministry of Health and Social Development of the Republic of Karelia and Petrozavodsk State University (Protocol 25 dated February 122013). We analyzed phenotypes of peripheral blood lymphocytes, as well as tumor-infiltrating lymphocytes (TILs) isolated from the clinical specimens of tumor tissue (n = 5) obtained from the patients with stage III CRC.
Adenosine is accumulated in the extracellular matrix in response to metabolic stress and cell breakdown, i.e. in ischemia, hypoxia, inflammation and injury. Therefore, we thought it would be interesting to study activation of CD4+CD39+ cells in the context of inflammation and immune suppression that bears no connection to oncogenesis. So, we recruited a comparison group consisting of 29 patients (mean age of 44.5 ± 18 years) with acute pancreatitis (AP). The diagnosis was established based on the classification accepted at the ninth All-Russian Congress of Surgeons in 2000. The inclusion criteria applied to the comparison group were: age from 18 to 70 years and acute pancreatitis. Patients with other co-morbidities, such as cancers and autoimmune disorders, were excluded from the study. Lymphocyte profiles were analyzed prior to treatment.
TILs were isolated by enzymatic disaggregation. Freshly explanted tissues were minced, placed into the medium for enzymatic disaggregation and incubated at room temperature for 2-3 hours. The medium was prepared from RPMI-1640 (PanEco, Russia) supplemented with 10% FBS (HyClone, USA), 100 |jg/ml gentamycin (Sigma, USA) and 1 mg/ml collagenase IV (PanEco, Russia). The obtained suspension was passed through sterile filters with 70- and 40-jm pores. Lymphocyte subpopulations were separated using 75% and 100% density gradients prepared from ficoll with a density of 1.077 g/cm3 (PanEco, Russia).
Expression of the studied molecules was measured by multicolor flow cytometry using Cytomics FC500 (Beckman Coulter, USA), monoclonal antibodies against CD4-FITC, CD8-FITC, CD25-PC5, and CD127-PC7 (Beckman Coulter, France); against CD3-PE, CD16-FITC, and CD19-FITC (Sorbent, Russia); against FOXP3-PE (eBioscience, USA); against CD39 (R&DSystems, USA), and the corresponding isotope controls. Intracellular expression of FOXP3 was analyzed using fixation and permeabilization buffers by eBioscience, USA. Expression of CD39 mRNA was measured by real-time PCR. Isolation and purification of nucleic acids were done using AxyPrep Blood Total RNA Miniprep Kit (Axygen, USA). CDNA synthesis was aided by random hexamer primers and M-MLV reverse transcriptase (Sileks, Russia). CDNA amplification and the analysis of amplification products conducted in real time were performed using a reagent mix containing the intercalating dye SYBR Green I (Evrogen, Russia) in iCycler Thermal Cycler (Bio-Rad, USA). To analyze the obtained data, we applied the 2-AACt method, where Ct was a threshold cycle and ACt was the difference between the values of threshold cycles for the reference (GAPDH) and target (CD39) genes. The expression level of the studied gene was calculated relative to the controls (healthy donors). Expression of the studied gene in the controls was taken as 1. The data were processed in Statistica 6.0; significance of differences between the groups was calculated using the Mann-Whitney U-test. Differences were considered significant at р < 0.05. To assess correlations between the variables, we used Spearman's rank correlation coefficient. The data are presented as M ± SD. The study was carried out on the equipment of the Shared Facility of the Federal Research Center Karelian Research Center of the Russian Academy of Sciences.
RESULTS
In the course of this work we measured the levels of CD4+CD39+ T cells in the peripheral blood and TILs of patients with CRC. We found that the number of CD4+CD39+ T cells in the peripheral blood varied a great deal both among diseased
and healthy individuals. The patients with advanced stages of CRC had significantly more CD4+CD39+ lymphocytes than the controls p < 0.05). At the same time, no significant differences in the CD4+CD39+ lymphocyte count were observed between the patients with stages I and II CRC (Fig. 1).
In the population of lymphocytes isolated from tumor specimens the number of CD4+CD39+ T cells was 4 times higher than in the peripheral blood of the same patients (Fig. 2A).
The proportion of CD4+CD39+ T cells was increased in the subpopulation of CD4+ T lymphocytes (Fig. 2B). CD39+ TILs also expanded in the subset of T cells that did not have the CD4 marker on their surface (Fig. 2C). At the same time, there were more CD4-CD39+ cells in the tumor tissue specimens than there were CD4+CD39+ (p < 0.05). This trend was not observed for the peripheral blood lymphocytes.
Previously, we studied a population profile of peripheral blood lymphocytes in patients with CRC, including T cells and their subsets, B cells and natural killer (NK) cells [19]. Cytometry findings are shown in Table 1.
The patients with CRC had fewer B cells than the controls, both in the early and advanced stages of the disease (p < 0.05). The levels of CD3+ T cells changed in stages III and IV CRC. In all CRC stages the patients had reduced levels of CD4+ T helpers (p < 0.05) and activated CD4+CD25+ T cells (p < 0.05); the levels of cytotoxic CD8+ lymphocytes (CTLs) were elevated (p < 0.05). No significant differences in the levels of NK cells were observed between the patients with CRC and the controls.
We established a few associations between the shifts in the population profile of lymphocytes and the number of CD4+CD39+ T cells in patients with CRC. Negative correlations were observed between the number of CD3+CD4+ T helpers and CD4+CD39+ T cells (r = -0.60, p < 0.05), between the
25 -|
20 -
15 -
10
5
number of CD3-CD19+ B cells and CD4+CD39+ T cells (r = -0.40, p < 0.05), and between the value of the immunoregulatory index (the CD4+ to CD8+ ratio) and the number of CD4+CD39+ T cells (r = -0.58, p < 0.05). Our findings suggest involvement of CD4+CD39+ T cells in the immune suppression during CRC formation.
Treg cells play an important role in oncogenesis. Expression of CD39 by Tregs and their participation in the synthesis of extracellular adenosine are believed to constitute one of the key mechanisms underlying the suppression of the immune response [15, 16]. In this study we measured the levels of Treg cells with the CD4+CD25+CD127lo/- phenotype. The patients with stages I and II CRC had increased levels of CD4+CD25+CD127lo/- Tregs in comparison with the healthy individuals, while in the patients with the advanced stages of CRC the number of these cells was the same as in the controls (Table 1). Tregs with the CD4+CD25+CD127lo/- and CD4+CD25hi phenotypes circulating in the peripheral blood of the patients with CRC increasingly expressed CD39 (Table 2).
As shown in Table 2, CD39 expression in CD4+CD25+CD127lo/- Tregs starts to go up in the very early stages of the disease (stages I and II), which does not happen in healthy controls, and reaches its maximum in patients with advanced cancer. The same pattern is observed for CD4+CD25hi Treg cells. In the cells with the CD4+CD25- phenotype, CD39 expression is quite low and does not differ in its intensity from that observed in the controls.
In addition, in this work we studied expression of the Treg transcription factor FOXP3 and its association with CD39 expression. We managed to establish a direct correlation between the expression of CD39 and the expression of FOXP3 in CD4+CD25hi T cells isolated from the peripheral blood of
p < 0,01
vw
Controls Patients with Patients with stages I stages III
and II CRC and IV CRC
Fig. 1. Levels of CD4+CD39+ T cells in the peripheral blood of patients with CRC
А
%
40 -30 -20 -10 -0
CD4+CD39+/lymphocytes
B
%
80 n 60 -40 -20 -0
CD4+CD39+/CD4+
C
%
60 1
CD4-CD39+/lymphocytes
Blood
Tumor
Blood
Tumor
Blood
Tumor
Fig. 2. Levels of CD39-expressing lymphocytes in the peripheral blood and tumor tissue of patients with CRR Note: * — represents significant differences at p < 0.05.
0
patients with CRC (r = 0.51, p < 0.05). The subset of CD4+CD25hi Tregs representing TILs also expressed CD39 more vigorously than peripheral blood lymphocytes. At the same time, almost all TILs with the CD4+CD25hi phenotype expressed CD39 (Table 2). However, increased CD39 expression was also observed in non-regulatory CD4+CD25- cells, as compared with the peripheral lymphocytes of the same patients. This suggests that cancer stimulates expression of CD39 in different subpopulations of CD4+ T cells, including Tregs.
Changes in the population profile of lymphocytes and the relative number of Tregs were also evaluated in patients with AP (Table 1). The levels of T lymphocytes, CD4+ T cells and activated T helpers were lower in these patients than in the controls. In contrast, CD25 expression by T helpers was higher in the patients with AP than in the controls (25.37 ± 8.6% and 18.09 ± 7.5% from the total number of CD4+ T cells, respectively, p < 0.05). Changing proportions of different T cell subsets manifested themselves as a drop in the value of the immunoregulatory index (IRI). Unlike healthy donors, the patients with AP had elevated levels of CD8+ CTLs and NK cells. Thus, the patients with AP, as well as the patients with CRC, showed signs of compromised immunity. Besides, the patients with AP had more peripheral CD4+CD25+CD127lo/-Treg cells than the controls (Table 1).
When studying the expression of the ectonucleotidase CD39, we found that in the patients with AP, CD4+CD39+ T cells made 9.16 ± 2.9% from the total count of CD4+ T cells. Expression of CD39 by Treg cells in the patients with AP followed a pattern similar to that observed for the patients with CRC. CD39 was increasingly expressed in the Tregs (p < 0.05) of the patients with AP. Expression of CD39 in the Treg cells with the CD4+CD25hi phenotype in the patients with AP was 57.98 ± 19.6%, while in CD4+CD25+CD127lo/- Tregs it amounted to 62.09 ± 16.4%. In CD4+CD25- lymphocytes that were not Tregs CD39 expression reached 7.67 ± 4.3% and was not reliably different from the levels observed in the controls.
We also studied involvement of CD4+CD39+ T cells in the immune suppression in the patients with AP. The correlation analysis of the CD4+CD39+ T cell count, the expression of
this molecule in CD4+CD25+CD127lo/- Tregs and the shifts in the population profile of lymphocytes in the patients with AP revealed no reliable associations, in contrast to the CRC situation.
Besides, we measured relative expression of CD39 mRNA in the peripheral blood leukocytes of patients with CRC and AP. MRNA expression was 2.36 times higher in the patients with CRC than in the controls (Fig. 3). No significant differences in the expression of CD39 transcripts were observed between the patients with AP and the controls.
DISCUSSION
Extracellular adenosine is a signal molecule that modulates many physiological processes in the body. Recently, adenosine-mediated suppression of the immune response has received a lot of attention as one of the key mechanisms helping cancer cells to evade the immune system. Adenosine is a product of adenosine monophosphate (AMP) dephosphorylation occurring in the extracellular matrix. One of the key enzymes involved in this process is the ectonucleotidase CD39; it ensures conversion of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to AMP [8, 9].
In the course of this work we explored the role of CD4+ T cells expressing CD39 in the formation of immune suppression in patients with CRC. We found that patients with CRC accumulate CD4+CD39+ cells in their peripheral blood in the advanced stages of cancer. Among TILs the number of CD4+ T cells expressing CD39 was significantly higher than in the blood of the same patients. Besides, the patients demonstrated a negative correlation between the levels of CD4+CD39+ T cells and a few other parameters, such as the levels of CD3+CD4+ T helpers, CD3-CD19+ B cells and the value of the immunoregulatory index, suggesting involvement of CD4+CD39+ T cells in immunosuppression during CRC progression.
Treg cells play an important role in promoting immune suppression in nascent and progressing cancer. Recently, it has been discovered that Tregs can engage in the accumulation of
Table 1. Percentage of major lymphocyte subsets In the peripheral blood of healthy donors, patients with CRC and patients with AP from the total number of lymphocytes
Phenotypes Controls Patients with CRC Patients with AP
stages I-II stages III-IV
CD3+ (T cells) 69.26 ± 5.3 66.96 ± 6.4 64.02 ± 6.4* 63.87 ± 7.1*
CD3+CD4+ (T helpers) 42.39 ± 6.4 32.80 ± 9.5* 35.66 ± 5.9* 34.46 ± 9.1*
CD4+CD25+ (activated T helpers) 10.74 ± 5.2 6.46 ± 2.7* 5.57 ± 1.8* 7.90 ± 3.2*
CD3+CD8+ (CTL) 21.98 ± 4.7 31.45 ± 6.1* 27.28 ± 6.5*# 28.20 ± 7.7*
CD3-CD19+ (B cells) 11.15 ± 3.0 8.13 ± 4.3* 5.99 ± 2.8* 7.90 ± 5.2
CD3-CD16+ (NK cells) 14.84 ± 5.7 13.39 ± 4.8 15.56 ± 8.5 17.65 ± 7.2*
CD4+/CD8+ (immunoregulatory index) 2.07 ± 0.5 1.12 ± 0.5* 1.40 ± 0.4* 1.36 ± 0.7*
CD4+CD25+CD127lo/- (Tregs) 4.56 ± 1.0 5.34 ± 1.9* 4.7 ± 1.4 6.83 ± 2.7*
Note: * — represents significant differences from the controls, p < 0.05; # — represents significant differences from patients with stages I and II CRC, p < 0.05. Table 2. Expression of ectonucleotidase C39 in the subset of CD4+ T cells isolated from patients with CRC presented as percentage from the total number of CD4+ T cells
CD4+CD25- CD4+CD25hl CD4+CD25+CD127lo/-
Controls 5.58 ± 3.9 42.70 ± 5.8 41.25 ± 2.7
Patients with stages I and II CRC 5.97 ± 3.6 53.85 ± 3.9* 55.32 ± 4.1*
Patients with stages III and IV CRC 7.54 ± 3.3 66.14 ± 3.4** 67.87 ± 2.9**
TIL 35.76 ± 22.6# 90.06 ± 7.1# Нет данных
Note: * — represents significant differences from the controls; ** — represents significant differences from the controls and patients with stages I and II CRC; # — represents significant differences from the peripheral blood lymphocytes of the same patients with CRC.
a
CL <
CD
£ ci
.33
2.5 -
2 -
1.5 -
1 -
0.5 -
Controls
AP
CRC
Fig. 3. Changes in the levels of CD39 mRNA In the peripheral blood leukocytes of patients with CRC and AP (relative to GAPDH mRNA) Note: * — represents significant differences from the controls at p < 0.05; data are presented as M ± SE.
0
extracellular adenosine. Unlike other T lymphocytes, these cells increasingly express CD39 required for their suppressive activity [20, 21]. It has been shown that Treg cells isolated from the blood of CD39-deficient mice exhibit a low level of suppressive activity in vitro and cannot prevent transplant rejection in vivo [22]. Murine CD4+CD25+ Tregs express on their surface both CD39 and CD73, the nucleotidase dephosphorylating AMP to adenosine. In humans, expression of CD73 by CD4+CD25hi T cells is very low [23], but in the cytoplasm it becomes more vigorous than in CD4+CD25- T lymphocytes [21]. Moreover, the cell culture of human Tregs has been shown to promote adenosine accumulation, confirming that these cells produce active CD73 molecules.
We were able to show that peripheral blood CD4+CD25hi and CD4+CD25+CD127lo/- T cells increasingly express CD39 in patients with colorectal cancer. High expression of this ectonucleotidase correlated with the stage of the disease and was also noted for CD4+CD25hi TILs. Given that, we conclude that TILs accumulate Tregs that increasingly express CD39+ as a result of cell recruiting from the peripheral pool of lymphocytes. These Tregs constitute one of the dominating subsets of Tregs in colorectal cancer, especially in its advanced stages. Besides, CD39+ Tregs exhibit a high immunosuppressive activity, which can promote cancer, along with other mechanisms of immune suppression.
Progression of severe AP is also accompanied by the changes in the reactivity of the immune system. AP is characterized by pancreatic inflammation that can affect peripancreatic tissue and lead to multiple organ failure occurring as a result of necrosis, infection or sepsis [24]. Presumably, overt immune manifestations of the systemic inflammatory response syndrome can trigger immunosuppression, leading to the inability of the body to resist microbial invasions, festering and necrotic complications [25].
Our findings about the changes in the population profile of lymphocytes and increased levels of Treg cells in patients with AP may be indicative of immune suppression occurring in such
patients. Considering elevated levels of Tregs, the inflammatory nature of the disease and the increasing apoptosis of circulating lymphocytes [26], it can be assumed that immune suppression in patients with AP is a compensatory mechanism restraining the inflammatory response.
Unlike patients with CRC, patients with AP had the same levels of CD4+CD39+ cells as the controls. No correlation has been observed between the proportions of major lymphocyte subsets and the levels of CD4+CD39+ T cells. In the patients with AP, expression of CD39 by Tregs (CD4+CD25hi and CD4+CD25+CD127lo/-) was higher than in the controls, which may be explained by the increased presence of Tregs in the blood of these patients. It is likely that in patients with AP CD4+CD39+ T cells do not make a considerable contribution to the development of systemic immune suppression as it happens in patients with cancer. This supposition is supported by the results of the analysis of relative CD39 mRNA levels in patients with CRC and AP. The patients with CRC have demonstrated a gradual increase in CD39 mRNA levels in the course of the disease, which reached its maximum in the advanced stages. At the same time, the patients with AP had the same levels of CD39 mRNA as the controls.
CONCLUSIONS
Progression of CRC is accompanied by the expansion of CD4+ T cells expressing CD39; active expansion of CD39+ cells is observed in the pool of TILs. These cells play an important role in the formation of immune suppression in patients with CRC. The substantial proportion of CD39-expressing cells is constituted by Treg lymphocytes. Inhibition of CD39 expression and/or restriction of Treg cell activity may be of interest for the development of new approaches to anticancer therapies. Further research is necessary to elucidate the mechanisms of adenosine-A2AR-mediated immune suppression in patients with cancer.
References
1. Kaprin AD, Starinski VV, Petrova GV. Sostoyanie onkologicheskoi 15. pomoshi naseliniyu Rossii v 2015. M.: MNIOI im. Gerchena; 2016.
250 s. Russian.
2. Tsimmerman YaS. Colorectal cancer: state-of-the-art. Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2012;
22 (4): 5-17. Russian. 16.
3. Mougiakakos D. Regulatory T cells in colorectal cancer: from biology to prognostic relevance. Cancers. 2011; 3: 1708-31. 17.
4. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, et al. Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J Clin Oncol. 2009;
27: 186-92. 18.
5. Nosho K, Baba Y, Tanaka N, Shima K, Hayashi M, Meyerhardt JA, et al. Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature 19. review. J Pathol. 2010; 222 (4): 4350-66.
6. Lutgens MW, Vleggaar FP, Schipper ME, Stokkers PC, van der Woude CJ, Hommes DW, et al. High frequency of early colorectal cancer in inflammatory bowel disease. Gut. 2008; 57: 1246-51. 20.
7. Lasry A, Zinger A, Ben-Neriah Y. Inflammatory networks underlying colorectal cancer. Nat Immunol. 2016; 17 (3): 230-40.
8. Antonioli L, Blandizzi C, Pacher P, Hasko G. Immunity, inflammation
and cancer: a leading role for adenosine. Nat Rev Cancer. 2013; 21. 13: 842-57.
9. Antonioli L, Pacher P, Vizi ES, Hasko G. CD39 and CD73 in immunity and inflammation. Trends Mol Med. 2013; 19: 355-67.
10. Sitkovsky MV, Kjaergaard J, Lukashev D, Ohta A. Hypoxia-adenosinergic immunosuppression: tumor protection by T 22. regulatory cells and cancerous tissue hypoxia. Clin Cancer Res. 2008; 14: 5947-52.
11. Jackson SW, Hoshi T, Wu Y, Sun X, Enjyoji K, Cszimadia E, et al. 23. Disordered purinergic signaling inhibits pathological angiogenesis
in cd39/Entpd1-null mice. Am J Pathol. 2007; 171: 1395-404.
12. Stagg J, Beavis PA, Divisekera U, Liu MC, Moller A, Darcy PK, et 24. al. CD73-deficient mice are resistant to carcinogenesis. Cancer
Res. 2012; 72: 2190-6. 25.
13. Beavis PA, Stagg J, Darcy PK, Smyth MJ. CD73: a potent suppressor of antitumor immune responses. Trends Immunol. 2012; 33: 231-7.
14. Bastid J, Cottalorda-Regairaz A, Alberici G, Bonnefoy N, Eliaou JF, 26. Bensussan A. ENTPD1/CD39 is a promising therapeutic target in oncology. Oncogene. 2013; 32: 1743-51.
Kravchenko PN, Zhulai GA, Churov AV, Oleinik EK, Oleinik VM, Barysheva OYu, i dr. Subpopulations of Regulatory T-lymphocytes in the Peripheral Blood of Patients with Rheumatoid Arthritis. Annals of the Russian Academy of Medical Sciences. 2016; 71 (2): 148-153. Russian.
Churov AV. Regulatory T cells and aging. Advances in gerontology. 2013; 26 (4): 603-609. Russian.
Morikawa H, Sakaguchi S. Genetic and epigenetic basis of Treg cell development and function: from a FoxP3-centered view to an epigenome-defined view of natural Treg cells. Immunological Reviews. 2014; 259 (1): 192-205.
Whiteside TL. Regulatory T cell subsets in human cancer: are they regulating for or against tumor progression? Cancer Immunol Immunother. 2014; 63: 67-72.
Zhulai GA, Oleinik EK, Romanov AA, Oleinik VM, Churov AV, Kravchenko PN. Circulating regulatory T-cells and changes in the subpopulation composition of lymphocytes in colorectal cancer patients. Problems in oncology. 2016; 62 (1): 96-100. Russian. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007; 204: 1257-65.
Mandapathil M, Hilldorfer B, Szczepanski MJ, Czystowska M, Szajnik M, Ren J, et al. Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells. Journal of Biological Chemistry. 2010; 285: 7176-86.
Ernst PB, Garrison JC, Thompson LF. Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology. J Immunol. 2010; 185: 1993-98.
Dwyer KM, Deaglio S, Gao W, Friedman D, Strom TB, Robson SC. CD39 and control of cellular immune responses. Purinergic Signal. 2007; 3: 171-80.
Al Mofleh IA. Severe acute pancreatitis: pathogenetic aspects and prognostic factors. World J Gastroenterol. 2008; 14: 675-84. Vinnik YuS, Cherdancev DV, Salmina AB, Markelova NM, Miller SV. Osobennosti regulyacii apoptoza immunokompetentnih kletok pri ostrom destruktivnom pankreatite. Novosti hirurgii. 2011; 9 (2): 37-42. Russian.
Zhang XP, Chen HQ, Liu F, Zhang J. Advances in researches on the immune dysregulation and therapy of severe acute pancreatitis. J Zhejiang Univ Sci B. 2009; 10 (7): 493-8.
Литература
1. Каприн А. Д., Старинский В. В., Петрова Г. В., редакторы. 9. Состояние онкологической помощи населению России в 2015. М.: МНИОИ им. Герцена; 2016. 250 с. 10.
2. Циммерман Я. С. Колоректальный рак: современное состояние проблемы. Российский журнал гастроэнтерологии, гепатологии, колопроктологии. 2012; 22 (4): 5-17.
3. Mougiakakos D. Regulatory T cells In colorectal cancer: from 11. biology to prognostic relevance. Cancers. 2011; 3: 1708-31.
4. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, et
al. Tumor-infiltrating FOXP3+ T regulatory cells show strong 12. prognostic significance in colorectal cancer. J. Clin. Oncol. 2009; 27: 186-92.
5. Nosho K, Baba Y, Tanaka N, Shima K, Hayashi M, Meyerhardt JA, 13. et al. Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature review. J Pathol. 2010; 222 (4): 4350-66. 14.
6. Lutgens MW, Vleggaar FP, Schipper ME, Stokkers PC, van der Woude CJ, Hommes DW, et al. High frequency of early colorectal cancer in inflammatory bowel disease. Gut. 2008; 57: 1246-51. 15.
7. Lasry A, Zinger A, Ben-Neriah Y. Inflammatory networks underlying colorectal cancer. Nat Immunol. 2016; 17 (3): 230-40.
8. Antonioli L, Blandizzi C, Pacher P, Haskô G. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer. 2013;
13: 842-57. 16.
Antonioli L, Pacher P, Vizi ES, Hasko G. CD39 and CD73 in immunity and inflammation. Trends Mol Med. 2013; 19: 355-67. Sitkovsky MV, Kjaergaard J, Lukashev D, Ohta A. Hypoxia-adenosinergic immunosuppression: tumor protection by T regulatory cells and cancerous tissue hypoxia. Clin Cancer Res. 2008; 14: 5947-52.
Jackson SW, Hoshi T, Wu Y, Sun X, Enjyoji K, Cszimadia E, et al. Disordered purinergic signaling inhibits pathological angiogenesis in cd39/Entpd1-null mice. Am J Pathol. 2007; 171: 1395-404. Stagg J, Beavis PA, Divisekera U, Liu MC, Moller A, Darcy PK, et al. CD73-deficient mice are resistant to carcinogenesis. Cancer Res. 2012; 72: 2190-6.
Beavis PA, Stagg J, Darcy PK, Smyth MJ. CD73: a potent suppressor of antitumor immune responses. Trends Immunol. 2012; 33: 231-7.
Bastid J, Cottalorda-Regairaz A, Alberici G, Bonnefoy N, Eliaou JF, Bensussan A. ENTPD1/CD39 is a promising therapeutic target in oncology. Oncogene. 2013; 32: 1743-51. Кравченко П. Н., Жулай Г. А., Чуров А. В., Олейник Е. К., Олейник В. М., Барышева О. Ю. И др. Субпопуляции регуляторных Т-лимфоцитов в периферической крови больных ревматоидным артритом. Вестник РАМН. 2016; 71(2): 148-153.
Чуров А. В. Регуляторные Т-клетки и старение организма.
Успехи геронтологии. 2013; 26 (4): 603-609.
17. Morikawa H, Sakaguchi S. Genetic and epigenetic basis of Treg cell development and function: from a FoxP3-centered view to
an epigenome-defined view of natural Treg cells. Immunological 22. Reviews. 2014; 259 (1): 192-205.
18. Whiteside TL. Regulatory T cell subsets in human cancer: are
they regulating for or against tumor progression? Cancer Immunol 23. Immunother. 2014; 63: 67-72.
19. Жулай Г. А., Олейник Е. К., Романов А. А., Олейник В. М., Чуров А. В., Кравченко П. Н. Циркулирующие регуляторные 24. Т-клетки и изменения в субпопуляционном составе лимфоцитов у больных колоректальным раком. Вопросы 25. онкологии. 2016; 62 (1): 96-100.
20. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, et al. Adenosine generation catalyzed by CD39 and CD73 expressed
on regulatory T cells mediates immune suppression. J Exp Med. 26. 2007; 204: 1257-65.
21. Mandapathil M, Hilldorfer B, Szczepanski MJ, Czystowska M, Szajnik M, Ren J, et al. Generation and accumulation of
immunosuppressive adenosine by human CD4+CD25hi9hFOXP3+ regulatory T cells. Journal of Biological Chemistry. 2010; 285: 7176-86.
Ernst PB, Garrison JC, Thompson LF. Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology. J Immunol. 2010; 185: 1993-98.
Dwyer KM, Deaglio S, Gao W, Friedman D, Strom TB, Robson SC. CD39 and control of cellular immune responses. Purinergic Signal. 2007; 3: 171-80.
Al Mofleh IA. Severe acute pancreatitis: pathogenetic aspects and prognostic factors. World J Gastroenterol. 2008; 14: 675-84. Винник Ю. С., Черданцев Д. В., Салмина А. Б., Маркелова Н. М., Миллер С. В. Особенности регуляции апоптоза иммунокомпетентных клеток крови при остром деструктивном панкреатите. Новости хирургии. 2011; 9 (2): 37-42. Zhang XP, Chen HQ, Liu F, Zhang J. Advances in researches on the immune dysregulation and therapy of severe acute pancreatitis. J Zhejiang Univ Sci B. 2009; 10 (7): 493-8.
