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Abstract

BACKGROUND:

The bone marrow immunosuppressive microenvironment of AML patients sustains and modulates proliferation, survival and drug resistance of AML through deregulation of both innate and adaptive immune response. We aimed to investigate the level of Tregs, expression of Tim-3 on peripheral blood T cells, expression of CD200 in myeloid blasts in newly diagnosed AML patients with normal cytogenetics (AML-NC) and their prognostic impact.

PATIENTS AND METHODS:

This study included 40 patients with de novo AML-NC and 20 healthy controls. Flow-cytometry was used for detection of CD4 $+$ CD25 $+$ high FoxP3 $+$ regulatory T cells, Tim-3 expression on peripheral blood T cells and CD200 expression on myeloid blasts.

RESULTS:

The percentages of CD4 $+$ CD25 $+$ high and CD4 $+$ CD25 $+$ high Foxp3 $+$ Tregs were significantly increased in AML patients than controls. The levels of Tregs, Tim-3/CD4 ${}^{+}$ , Tim-3/CD8 ${}^{+}$ , CD200 and MFI of CD200 were significantly lower in responding patients than in those with persistent leukemia. Only high CD200 expression ( $>$ 50%) showed statistically significant worse OS with $P<$ 0.04.

CONCLUSION:

The increased levels of Tregs, Tim-3 expression on peripheral blood T cells and CD200 expression in myeloid blast in AML patients could play a role in the development of AML. Analysis of these markers could serve as prognostic markers and might guide the therapy in AML patients in the future.

Keywords

Regulatory T cells CD200 TIM3 acute myeloid leukemia normal cytogenetics

1. Introduction

Acute myeloid leukemia (AML) represents a group of clonal hematopoietic stem cell disorders that result from genetic alterations in normal hematopoietic stem cells [1]. About 40% of AML patients are classified as intermediate risk without identifiable cytogenetic abnormalities. Patient outcomes in AML with normal cytogenetics (AML-NC), in particular, are widely diverse [2]. Molecular markers such as FLT3, NPM1, and CEBPA mutations have added a great deal to the prognostic stratification of AML-NC. However, it is vital to build upon these advances by continuing to expose the biological characteristics and properties of leukemic stem cells and their regulating factors to reach optimum AML treatment plans [3].

AML microenvironment is immunosuppressive and anti-apoptotic facilitating the survival of malignant hematopoietic cells. In preclinical model, AML cells secrete soluble factors, which limit proin?ammatory T helper-1 cytokine production and inhibit T cell activation and proliferation [4]. However, this effect is reversed when regulatory T cells (Tregs) and other T lymphocytes are removed from the microenvironment, resulting in augmented immune responses to AML [5]. Tregs are a group of CD4 ${}^{+}$ T cells comprising about 10% of the normal CD4 ${}^{+}$ T-cell counts. Tregs characterized by the expression of fork head box transcription factor P3 (Foxp3), and high level of CD25. Increased proportions and/or functional activities of Tregs are described in different solid and hematological malignancies [6] [7].

CD200 is a type-1a transmembrane cell-surface glycoprotein which is expressed normally in some tissues such as the central nervous system and testis, and certain leukocytes, including T and B lymphocytes, where its role is to protect immune-privileged sites and promote peripheral tolerance [8]. CD200 has no known intracellular signaling motif, but induces immunosuppression by binding with CD200R, a cell-surface receptor homolog, which is expressed on certain T-cell population and leukocytes of myeloid lineage including mast-cells, macrophages, basophiles, dendritic cells (DCs) and this binding favors the tumor growth [9]. CD200 has the potential to induce the formation of Tregs [10]. Diagnostic and prognostic role of CD200 has been demonstrated in different hematological malignancies as in chronic lymphocytic leukemia and multiple myeloma [11].

In addition, CD200 had a negative prognostic impact in cytogenetically – normal AML; patients with high expression of CD200 had a significantly lower 3-year DFS and 3-year OS [12].

T cells immunoglobulin and mucin domain 3 (Tim-3), which belongs to TIMs family is expressed by IFN- $\gamma$ – secreting T-helper 1 (Th1) cells and subsequently on DCs, monocytes, CD8 ${}^{+}$ T cells, and other lymphocyte subsets. Tim-3 binds to its potential ligand, galectin-9 thus inhibiting Th1 responses and inducing peripheral tolerance [13]. Tim-3, along with cytotoxic T lymphocyte antigen 4 (CTLA-4) and Programmed Death 1(PD-1), comprise what is called immune checkpoint molecules. Several studies emphasized the high expression of Tim-3 on tumor infiltrating T cells of many solid tumors, and this high expression of Tim-3 was associated with tumor genesis “tumor permissive” and progression [14].

Still, little is known about the role of Tregs, Tim-3 and CD200 expression in AML. Our aim was to investigate the level of Tregs, the expression of Tim-3 on peripheral blood T cells, the expression of CD200 on myeloid blasts in newly diagnosed AML-NC patients and the relation between these markers. Also we evaluated their prognostic impact on treatment response and outcome of the disease.

2. Patients and methods

This study was a prospective case – controlled study included 40 patients with de novo AML-NC presented to South Egypt Cancer Institute (SECI), Assiut University during the period from January 2014 to June 2016. AML M3 patients were excluded due to different disease biology and treatment strategies. Twenty age and sex matched health controls were also included in the study. The study was approved by the Institutional Review Board of the SECI, Assiut University. An informed written consent was taken from all patients and controls.

All patients and controls were subjected to thorough history taking and clinical examination, with careful assessment of clinical signs relevant to leukemia as hepatomegaly, splenomegaly, lymphadenopathy, and gum or skin infiltration. Complete blood pictures were performed by the fully automated blood counters.

The patients only were subjected to

•
Bone marrow examination and cytochemistry, such as myeloperoxidase, esterases, acid phosphatase and Periodic acid-Schiff.
•
Flow cytometric immunophenotyping using monoclonal antibodies that were used for diagnosing AML includes: CD34, CD13, CD33, CD117, CD15 and intracellular myeloperoxidase, CD14, HLA-DR, CD41, CD61 and anti-glycophorin A. All monoclonal antibodies were purchased from Becton Dickinson (BD) Bioscience, CA, USA.
•
Flow cytometric detection of the Tim-3 expression on peripheral blood T cells.
•
Cytogenetic studies included conventional banding karyotyping and FISH for t(8,21), inv16 and t(15,7).
•
Flow cytometric detection of CD200 expression on myeloid blasts.

Figure 1.
Flow cytometric detection of regulatory T cells. A: Forward and side scatter histogram was used to define the lymphocytes population (R1). B: The expression of CD4 in the lymphocytes population was detected; thenCD4 ${}^{+}$ cells were gated for further analysis of CD25. C: Different gates were drowning to define CD4 ${}^{+}$ CD25 ${}^{-}$ cells, CD4 ${}^{+}$ CD25 ${}^{+\text{low}}$ cells and CD4 ${}^{+}$ CD25 ${}^{+\text{high}}$ cells. D: The percentage of CD4 ${}^{+}$ CD25 ${}^{+\text{high}}$ Foxp3 ${}^{+}$ cells (regulatory T cells) was then detected.

The diagnosis was based on standard morphologic, cytochemical, immunophenotypic, and cytogenetic criteria. After conventional induction therapy with 3 days of an anthracycline and 7 days of cytarabine (“3 $+$ 7”), response assessment is commonly performed between day 21 and day 28 after start of therapy. Overall survival (OS) was calculated from date of diagnosis to date of death (irrespective of the cause). Response to treatment was defined according to the revised recommendations from the international expert panel, on behalf of the European Leukemia Net on diagnosis and management of acute myeloid leukemia in adults [15].
2.1 Flow cytometric detection of CD4 ${}^{+}$ CD25 ${}^{+}$ high FoxP3 ${}^{+}$ regulatory T cells

CD4 ${}^{+}$ CD25 ${}^{+}$ high Foxp3 ${}^{+}$ Tregs in whole blood samples were enumerated using phycoerythrin (PE) conjugated anti CD25 (IQ Product the Netherland), fluoroisothiocyanate (FITC) conjugated anti Foxp3 (Bioscience, USA) and Peridinium-chlorophyll-protein (Per-CP)-conjugated anti CD4 (BD Bioscience, CA, USA). Fifty $\mu$ l of blood sample was incubated with 5 $\mu$ l of anti CD4, anti CD25 for 15 minutes at 4 ${}^{\circ}$ C in the dark. Following incubation, red blood cells lysis, washing with phosphate buffer saline (PBS), addition of fixed solution to fix the cells and incubation for 10 minutes were done. After incubation, cells were washed with PBS, and 5 $\mu$ l of anti Foxp3 were added and incubated for 20 minutes at 4 ${}^{\circ}$ C. After one wash, the cells were re-suspended in PBS, and analyzed by FACSCalibur flow cytometry with Cell Quest software (BD Biosciences, USA). An isotype-matched negative control was used for each sample. Forward and side scatter histogram was used to define the lymphocytes population (R1). Then CD4 $+$ cells were gated. Total CD4 ${}^{+}$ CD25 ${}^{+}$ , CD4 ${}^{+}$ CD25 ${}^{+}$ low, CD4 ${}^{+}$ CD25 ${}^{+}$ High (defined as the population of CD4 positive T cells whose CD25 expression exceeded the level of CD25 positivity seen in the CD4 negative T cells) and CD4 ${}^{+}$ CD25 ${}^{+}$ High Foxp3 ${}^{+}$ Tregs was evaluated as a percentage of CD4 $+$ cells as shown in Fig. 1. The expression of Foxp3 $+$ in CD4 ${}^{+}$ CD25 ${}^{+}$ high cells was expressed as geometric mean of fluorescence intensity (MFI).

Figure 2.

Flow cytometric detection of the Tim-3 expression on peripheral blood T cells. A: Forward and side scatter histogram was used to define the lymphocytes population (R1). B: The expressions of CD4 and CD8 in the lymphocytes population were detected, and then CD4 ${}^{+}$ cells and CD8 ${}^{+}$ cells were gated for further analysis of TIM3. C: The expression of TIM3 in CD8 ${}^{+}$ cells. D: The expression of TIM3 in CD4 ${}^{+}$ cells.

2.2 Flow cytometric detection of the Tim-3 expression on peripheral blood T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll density gradient centrifugation (Biochrom GmbH, Germany). The cells were washed and 2 $\times$ 106 cells were stained with FITC anti-Tim-3 (Biolegend GmbH, Germany), PE- anti- CD8 (BD Bioscience, CA, USA), Per-CP anti-CD4 (BD Bioscience, CA, USA) for 20 min on ice. After one wash, the cells were re-suspended in PBS, and analyzed by FACSCalibur flow cytometry with Cell Quest software (BD Biosciences, USA). An isotype-matched negative control was used for each sample. Forward and side scatter histogram was used to define the lymphocytes population. Then CD4 ${}^{+}$ cells and CD8 ${}^{+}$ cells were gated. Then the expression of Tim-3 on CD4 ${}^{+}$ cells (Tim-3/CD4 ${}^{+}$ ) cells and on CD8 ${}^{+}$ cells (Tim-3/CD8 ${}^{+}$ ) was detected. The results was expressed as percentage of CD4 ${}^{+}$ and CD8 ${}^{+}$ cells as shown in Fig. 2.

2.3 Flow cytometric detection of the CD200 expression on myeloid blasts

Fifty $\mu$ l of bone marrow sample was incubated with 5 $\mu$ l of FITC- anti- CD200, PE- anti CD33 and Per-CP anti CD34 (all from BD Biosciences, CA, USA) for 20 at 4 ${}^{\circ}$ C in the dark. Following incubation, red blood cells lysis and washing with PBS were done. After washing, the cells were re-suspended in PBS, and analyzed by FACSCalibur flow cytometry with Cell Quest software (BD Biosciences, USA). An isotype-matched negative control was used for each sample. Forward and side scatter histogram was used to define the blast cell population. The blast cells were gated for further analysis of the expression of CD34 and CD33. Then the expression of CD200 was detected in CD34 ${}^{+}$ CD33 ${}^{+}$ cells (myeloid blasts). CD200 expression evaluated as percentage of myeloid blasts (Fig. 3). The blasts was classified as either myeloid blasts with CD200low in which the percentage of expression of CD200 from ( $<$ 50%), and CD200high in which the percentage of expression of CD200 $>$ 50% of gated myeloid blast cells [16].

Figure 3.

Flow cytometric detection of the CD200 expression on myeloid blasts. A: Forward and side scatter histogram was used to define the blast cells (R1). B and C: The expressions of CD33 and CD34 were assessed in blast cells to identify myeloid blasts which are then gated for further expression of CD200. D: The expression of CD200 as a geometric mean of fluorescence intensity (MFI). The positivity was defined as fluorescence (open histogram) higher than that of the isotype control (purple histogram).

3. Statistical analysis

Statistical analysis was carried out using SPSS statistical software version 18. Qualitative data are expressed as frequency and percentage; quantitative data are expressed by mean $\pm$ standard error (SEM). Mann-Whitney analysis was used to detect the statistical significance differences between groups. Spearman’s correlation was used to correlate the studied parameters. $P$ value $<$ 0.05 was considered significant. Factors affecting CR were assessed by multivariate logistic regression, and expressed as HR (95% CI). Survival rates were calculated according to Kaplan-Meier analysis, differences among groups calculated by log-rank test. Chi square statistics was used to assess the prognostic significance of survival and significance between two subgroups of various parameters.

4. Results

The baseline characteristic of the patients and controls were presented in Table 1. The white blood cells count was significantly increased in patients than controls. Platelets count and hemoglobin concentration were significantly decreased in patients than the controls (Table 1). FAB types distribution was as follow; M2 was the most common FAB type (33%) followed by M4 (22%), while M1 and M5 had equal distributions of 12% each.

Table 1
Some characteristics of AML-NC patients and the controls

	Patients (40)	Controls (20)	$P$ -value
Age	35 $\pm$ 2.9	32 $\pm$ 1.72	–
Sex (male/female)	14/6	13/7	–
WBCs (10 ${}^{9}$ /L)	23.96 $\pm$ 4.3	7.63 $\pm$ 0.25	$<$ 0.001
Platelets (10 ${}^{9}$ /L)	42.65 $\pm$ 4.95	216.20 $\pm$ 14.58	$<$ 0.001
Hemoglobin (gm/dl)	7.43 $\pm$ 0.34	12.56 $\pm$ 0.26	$<$ 0.001
Bone marrow blast (%)	52.52 $\pm$ 2.95	–	–
Peripheral blood blast (%)	24.52 $\pm$ 1.74	–	–

Mann-Whitney Test. Data represented as means $\pm$ SEM, $P\leqslant$ 0.05 is significant, WBCs: white blood cells.

Table 2

Lymphocytes, regulatory T cells and expression of Tim-3 on the peripheral T lymphocytes in AML-NC patients and the controls

	Patients (40)	Control (20)	$P$ value
Lymphocytes (%)	41.13 $\pm$ 2.4	56.32 $\pm$ 2.8	$<$ 0.001
CD4 ${}^{+}$ (%)	26.01 $\pm$ 1.22	36.54 $\pm$ 2.81	0.012
CD8 ${}^{+}$ (%)	11.99 $\pm$ 1.5	20.54 $\pm$ 1.72	$<$ 0.001
CD4 ${}^{+}$ CD25 ${}^{+}$ /CD4 ${}^{+}$ (%)	19.43 $\pm$ 0.61	18.80 $\pm$ 0.51	0.371
CD4 ${}^{+}$ CD25 ${}^{\text{low}}$ /CD4 ${}^{+}$ (%)	14.69 $\pm$ 0.43	15.66 $\pm$ 0.41	0.194
CD4 ${}^{+}$ CD25 ${}^{+\text{high}}$ /CD4 ${}^{+}$ (%)	5.47 $\pm$ 0.46	4.34 $\pm$ 0.21	0.012
Tregs (%)	1.42 $\pm$ 0.15	1.06 $\pm$ 0.07	0.039
MFI of Foxp3 ${}^{+}$ expression in CD4 ${}^{+}$ CD25 ${}^{+\text{High}}$	109.25 $\pm$ 5.5	116.20 $\pm$ 12.1	0.552
Tim-3 $+$ /CD4 ${}^{+}$ (%)	6.03 $\pm$ 0.31	0.91 $\pm$ 0.05	$<$ 0.001
Tim-3 $+$ /CD8 ${}^{+}$ (%)	7.09 $\pm$ 0.51	1.07 $\pm$ 0.05	$<$ 0.001

Mann-Whitney Test. Data represented as means $\pm$ SEM. MFI: mean florescence intensity; Tim-3: T cells immunoglobulin mucin 3; Tregs: regulatory T cells.

Table 3

Correlations between regulatory T cells and expression of Tim-3 on T lymphocytes and CD200 in AML-NC patients and other prognostic factors

		Tregs	Tim-3 $+$ /CD4	Tim-3 $+$ /CD8	CD200	BM Blast	PB Blast	CD34	CD56	CD4	CD8	HB	PLT count	WBC
r	Tregs	1	0.554	0.653	0.524	0.421	0.268	0.541	0.524	$-$ 0.455	$-$ 0.325	0.292	$-$ 0.251	0.231
$P$ value			0.000	0.000	0.001	0.006	0.094	0.001	0.001	0.037	0.023	0.212	0.118	0.075
r	Tim-3 $+$ /CD4	0.554	1	0.893	0.385	0.662	0.507	0.318	0.385	$-$ 0.477	$-$ 0.308	0.318	$-$ 0.258	0.216
$P$ value		0.000		0.000	0.014	0.000	0.001	0.028	0.014	0.000	0.041	0.056	0.108	0.118
r	Tim-3 $+$ /CD8	0.653	0.893	1	0.347	0.302	0.090	0.434	0.359	$-$ 0.520	$-$ 0.441	0.152	$-$ 0.202	0.208
$P$ value		0.000	0.000		0.028	0.058	0.579	0.039	0.028	0.000	0.043	0.218	0.154	0.198
r	CD200	0.524	0.385	0.347 ${}^{\ast}$	1	0.360	0.267	0.347	0.398	$-$ 0.394	$-$ 0.225	0.106	0.055	0.227
$P$ value		0.001	0.014	0.028		0.023	0.096	0.028	0.048	0.046	0.163	0.517	0.735	0.149

Spearman’s correlation. r $=$ correlation coefficient; Tregs: regulatory T cells; Tim-3: T cells immunoglobulin mucin 3; HB: hemoglobin.

Table 4

Lymphocytes, regulatory T cells, expression of Tim-3 on the peripheral T lymphocytes and the expression of CD200 on myeloid blasts in AML-NC patients who achieved complete remission and who did not achieve complete remission

	Patients with CR (26)	Patients without CR (14)	$P$ value
Lymphocytes (%)	39.96 $\pm$ 2.1	43.67 $\pm$ 2.5	0.305
CD4 ${}^{+}$ (%)	27.04 $\pm$ 1.2	21.79 $\pm$ 1.7	0.005
CD8 ${}^{+}$ (%)	11.82 $\pm$ 1.5	14.98 $\pm$ 1.12	0.076
Tregs (%)	1.22 $\pm$ 0.12	1.95 $\pm$ 0.34	0.041
MFI of Foxp3 ${}^{+}$ expression in CD4 ${}^{+}$ CD25 ${}^{+\text{High}}$	118.16 $\pm$ 6.7	102.89 $\pm$ 1.5	0.511
Tim-3 $+$ /CD4 ${}^{+}$ (%)	5.28 $\pm$ 0.34	6.91 $\pm$ 0.41	0.012
Tim-3 $+$ /CD8 ${}^{+}$ (%)	6.47 $\pm$ 0.61	9.75 $\pm$ 0.75	0.005
CD200 (%)	34.95 $\pm$ 3.6	72.62 $\pm$ 4.41	0.001
MFI of CD200 expression	94.61 $\pm$ 10.9	157.2 $\pm$ 19.56	0.004

Mann-Whitney Test. Data represented as means $\pm$ SEM. MFI: mean florescence intensity, Tim-3: T cells immunoglobulin mucin 3; CR: complete remission, Tregs: regulatory T cells.

As shown in Table 2, the percentages of total lymphocytes, T helper cells (CD4 ${}^{+}$ ) and cytotoxic T (CD8 ${}^{+}$ ) lymphocytes were significantly decreased in AML patients than the controls (41.13 $\pm$ 2.4, 26.09, 11.99 vs. 56.32 $\pm$ 2.8, 36.54 and 20.54 respectively). The percentages of total CD4 ${}^{+}$ CD25 ${}^{+}$ /CD4 ${}^{+}$ and CD4 ${}^{+}$ CD25 ${}^{+\text{low}}$ /CD4 ${}^{+}$ were not significantly different between AML patients and the controls. However, the percentages of CD4 ${}^{+}$ CD25 ${}^{+\text{High}}$ /CD4 ${}^{+}$ and CD4 ${}^{+}$ CD25 ${}^{+\text{High}}$ Foxp3 ${}^{+}$ Tregs were significantly increased in AML patients than the controls (5.47 $\pm$ 0.46 vs. 4.34 $\pm$ 0.21 $P<$ 0.012. 1.42 $\pm$ 0.15 vs. 1.06 $\pm$ 0.07 $P<$ 0.039). Higher Tim-3 expression on helper T cells (Tim-3+/CD ${}^{4+}$ ) and cytotoxic T cells (Tim-3+/CD8 ${}^{+}$ ) were significantly increased in AML patients than the controls (6.03 $\pm$ 0.31 vs. 0.91 $\pm$ 0.05 $P<$ 0.001) and (7.09 $\pm$ 0.51 vs. 1.07 $\pm$ 0.05 $P<$ 0.001) respectively (Table 2).

CD200 expression was found in 26/40 patients (65%). The mean percentage of CD200 expression on myeloid blast of AML patients was 46.37 $\pm$ 4.6 and the MFI of CD200 expression was 79.35 $\pm$ 9.4. Twenty three patients (57.5%) had high expression of CD200 compared to 17 patients (42.5%) had CD200 low expression.

There were significant positive correlations between the Tregs and Tim-3 $+$ /CD4 ${}^{+}$ , Tim-3+/CD8 ${}^{+}$ and CD200 expression on myeloid blasts with each other. There were also significant positive correlations between them and some prognostic factors as myeloid expression of CD34 and CD56 and the percentage of bone marrow blast cells (Table 3). There was no significant correlations between the levels of Tregs, Tim-3/CD4 ${}^{+}$ and Tim-3/CD8 ${}^{+}$ , CD200 expression and other characteristics studied in AML-NC patients as percentages of CD4 $+$ and CD8 $+$ , hemoglobin level, platelet count and white blood cell count.

Table 5

Multivariate analysis of factors for CR

	HR (95% CI)	$P$ value
Age $\geqslant$ 50 yrs	1.86 (0.30–3.69)	0.45
High WBC ( $>$ 30.000/mm ${}^{3}$ )	0.92 (0.61–1.39)	0.78
CD34 positivity	2.45 (0.29–4.89)	0.65
CD200 expression ( $>$ 50%)	0.43 (0.25–0.81)	0.04*

Logistic regression Test. WBC: White blood cell, Tim-3: T cells immunoglobulin mucin 3, CR: complete remission, Tregs: regulatory T cells.

65% (26/40) of our patients achieved complete remission (CR). The percentages of Tregs, Tim-3/CD4 ${}^{+}$ , Tim-3/CD8 ${}^{+}$ , CD200 and MFI of CD200 were significantly lower in AML patients who achieved CR than those with persistent leukemia. CD4 ${}^{+}$ cell percentage was significantly higher in AML patients who achieved CR than in those with persistent leukemia, while there was no significant difference in total lymphocytes, cytotoxic T lymphocytes and MFI of Foxp3 ${}^{+}$ expression in CD4 ${}^{+}$ CD25 ${}^{+}$ between patients with CR and those with less than CR (Table 4). In multivariate analysis of prognostic factors for response including age ( $\geqslant$ 50 years), WBC count ( $>$ 30.000/mm ${}^{3}$ ), CD34 positivity and high CD200 expression ( $>$ 50%); only high CD200 expression ( $>$ 50%) remained to be associated with low likelihood of achieving CR (HR $=$ 0.43, 95% CI: 0.25–0.81, $P<$ 0.04) (Table 5). As regarding the prognostic factors of OS, only high CD200 expression ( $>$ 50%) showed statistically significant worse effect on the OS; $P=$ 0.04 (Fig. 4). None of Tregs level, Tim-3/CD4 ${}^{+}$ or Tim-3/CD8 ${}^{+}$ expression had an impact on OS with $P=$ 0.12, 0.11 and 0.11 respectively.

Figure 4.

Overall survival of the entire population according to CD200 expression. The blasts was classified as either myeloid blasts with CD200low in which the percentage of expression of CD200 from ( $<$ 50%), and CD200high in which the percentage of expression of CD200 ( $>$ 50%) of gated myeloid blast cells.

5. Discussion

Despite remarkable advances in understanding the molecular pathogenesis of AML during the past few years, the prognosis is still dismal in adults and elderly population [17].Over several decades, hitherto monolithic approaches “3 $+$ 7 protocol” is still the backbone chemotherapy. This is due, at least in part, to the heterogeneity of AML and lack of knowledge about full biological characteristics of the disease [17, 18].

The immune milieu interaction with AML cells is one of many factors attributing to disease resistance or relapse with worse outcome [18]. AML employ a number of immune evasion mechanisms which simulate “immune permissive” background. For example, poor T cell co-stimulation by tumor cells leading to T cell anergy, expression of negative co-stimulatory ligands such as PD-L1 and Galectin-9, production of immune suppressive cytokines and enzymes, expansion and/or induction of Tregs cells [19]. In the present study, we found higher levels of Tregs in the peripheral blood of AML patients compared to the healthy controls, a finding consistent with several previous reports [20]. Szczepanski et al. found higher CD4 ${}^{+}$ CD25 ${}^{\text{high}}$ Tregs percentage in the AML patients (4.5 $\pm$ 0.2% vs. 1.5 $\pm$ 0.08%, $P<$ 0.0001) compared with controls [19]. The increased level of Tregs in our study patients could imply that Tregs may play a role in the pathogenesis of AML. Tregs have a key role in suppressing T-cell-mediated immunity in animal models, hence tumor progression [7].

Regarding the impact of Tregs on treatment outcome of AML, Tregs frequency in the peripheral blood of AML patients at presentation appeared to correlate negatively with response to induction chemotherapy but not OS of patients. These findings indicate that the higher Tregs is associated with poor prognosis of AML, which is comparable with results reported by others [20]. For example, Szczepanski et al. found that patients who achieved CR had lower CD4 ${}^{+}$ CD25 ${}^{\text{high}}$ Treg at diagnosis than patients with persistent leukemia after induction chemotherapy (4.1 $\pm$ 0.3% vs. 5.6 $\pm$ 0.6%; $P<$ 0.04). Similarly, the CD4 ${}^{+}$ Foxp3 ${}^{+}$ population was lower (3.8 $\pm$ 0.4% vs. 5.2 $\pm$ 0.6%) in these AML responding patients [19]. Fortunately enough, this might have clinical implications. The inhibition of Tregs and blockade of CTLA-4 enhances tumor immunity in animal models of cancer [21]. On clinical level, blocking of CTLA-4 by monoclonal antibodies can shift the immune system balance toward T-cell activation, resulting in tumor rejection by the host. Hence, the anti-CTLA-4 blocking antibody ipilimumab was recently approved by the Food and Drug Administration for the treatment of melanoma [22].

We found an aberrant expression of CD200 in 65% of cases, with a high intensity of expression in 57.5% of AML patients. Also patients with CD200 over expression in leukemic cells have elevated level of Tregs. This was in agreement with Coles et al. They showed that CD200 blast expression level correlated significantly with the frequency of Tregs cells. CD200 had a direct effect on Tregs induction through engagement with CD200R on T cells in AML [10].These results are comparable with what confirmed by others [18, 23]. Memarian et al. found that CD200, but not CD200R, had elevated expression in AML myeloid progenitor cells compared to normal subjects [23].

While we did not find differences in CD200 expression among FAB subtypes, Tonks et al. found differential expression especially among complicated cytogenetics. There was a high frequency of CD200 expression in patients with t(8,21) leukemias. These patients also significantly over expressed CD200 compared to FAB-M2 patients without this cytogenetic abnormality. Furthermore, patients with inv(16) mutation (generally associated with FAB-M4) significantly over expressed CD200 when compared to M4 patients without an inv(16) mutation [24].

In our study, AML patients with high expression of CD200 were shown to have bad response to therapy. In addition, AML patients with CD200 high expression were found to have inferior OS compared to those patients with CD200 low expression. This was comparable to the results reported by Damian et al. where they observed a negative impact on OS in CD56 negative AML patients. Thoroughly, In CD56- AML, CD200 expression was associated with a lower survival probability; 3-year OS was 57% in CD200 negative, 35% in CD200 low and 0% in CD200 high patients. Conversely, no survival difference was evident regarding CD200 and CD34 expression [24]. Similarly, it was in agreement with the studies by Atfy and Tonks et al. [16, 24] respectively. Once again, this might have clinical implications. Two anti-CD200 antibodies have confirmed pre-clinical efficacy and could show therapeutic benefits in chronic lymphocytic leukemia and allograft survival of organ transplantation [26].

Tim-3 T-lymphocytes were a poor prognostic factor. In our study, the expression of Tim-3 on helper T lymphocytes (Tim-3/CD4 ${}^{+}$ ) and cytotoxic T lymphocytes (Tim-3/CD8 ${}^{+}$ ) were significantly increased in AML patients than the controls. Also there were positive correlations between Tim-3/CD4 ${}^{+}$ and Tim-3/CD8 ${}^{+}$ and blast cells and the expression of CD34 and CD56. In addition to that, the levels of Tim-3/CD4 ${}^{+}$ and Tim-3/CD8 ${}^{+}$ in patients who had achieved CR were lower compared to those with persistent leukemia as previously confirmed by Li et al. Furthermore, they documented that Tim-3 expression on CD4 ${}^{+}$ and CD8 ${}^{+}$ T cells was correlated with FLT3-ITD mutation and AML high risk respectively [27].

There is a hierarchical process of T cell exhaustion and diverse inhibitory receptors which regulate this incapacitation. Galectin-9 interaction, Tim3+PD1-, and CTLA-4 are some of these interdigitating processes [28, 29, 30]. Similarly, we found the same interrelated results. The positive correlations between Tregs, Tim-3 expression on peripheral blood T cells and the expression of CD200, and their negative correlations with CD4 ${}^{+}$ T cells and CD8 ${}^{+}$ T cells that were found in our results indicate that the immune system in AML patients is a complicated system in which all immune cells influence and control each other.

This study spotlights on several immune deregulations in case of AML-NC that could contribute to disease biology with poor outcomes. However, correlation with molecular markers of AML is not studied so as to reach firm disease prognostication based on these immune aberrations.

6. Conclusion

The increased levels of Tregs, Tim-3 expression on peripheral blood T cells and CD200 expression in myeloid blast in patients with AML could play a role in the development of AML. Analysis of Tregs, Tim-3 expression and CD200 expression could serve as prognostic markers and might guide the therapy in AML patients in the future.

Footnotes

Conflict of interest

The authors declared that they had no conflict of interest.

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Up-regulation of regulatory T cells,CD200 and TIM3 expression in cytogenetically normal acute myeloid leukemia

Abstract

BACKGROUND:

PATIENTS AND METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Patients and methods

2.3 Flow cytometric detection of the CD200 expression on myeloid blasts

4. Results

Table 1 Some characteristics of AML-NC patients and the controls

6. Conclusion

Footnotes

Conflict of interest

References

Table 1
Some characteristics of AML-NC patients and the controls