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Blood Res (2024) 59:42

Published online December 18, 2024

https://doi.org/10.1007/s44313-024-00048-0

© The Korean Society of Hematology

Increased IDO expression and regulatory T cells in acute myeloid leukemia: implications for immune escape and therapeutic targeting

Raziyeh Hakak1, Behzad Poopak1,2* and Ahmad Majd1

1 Department of Cellular and Molecular Biology, Faculty of Biological Sciences, North Tehran Branch, Azad University, Tehran, Iran
2 Payvand Clinical and Specialty Laboratory, Tehran, Iran

Correspondence to : Behzad Poopak
bpoopak@gmail.com

Received: September 14, 2024; Accepted: December 4, 2024

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Abstract

Purpose This study aimed to determine the frequency of regulatory T cells (Tregs) (CD4+/FOXP3+) and indoleamine 2,3-dioxygenase (IDO) expression in patients with acute myeloid leukemia (AML).
Methods This cross-sectional case–control study was conducted between Jan 2022 and Dec 2023. Bone marrow samples were collected from 20 healthy individuals and 15 patients with AML. Flow cytometry, real-time polymerase chain reaction (PCR), and western blotting were used to evaluate the frequency of Treg and IDO expression levels.
Results The Treg percentage among total lymphocytes was lower in the AML group than that in the normal group. However, Treg percentage among T-helper (Th) lymphocytes was significantly higher in the AML group than that in the normal group (p < 0.05). The mean IDO expression in the AML group was significantly higher than that in the normal group (p = 0.004). A significant relationship was observed between IDO expression and Treg percentage among Th lymphocytes in the AML group (correlation = 0.637; p = 0.003). Moreover, western blot analysis showed a significant increase in IDO protein intensity in the AML group compared with that in the control group (p < 0.001). A significant difference was observed between the IDO concentrations in the AML group and that in the control group (p < 0.001). In addition, a significant difference between TGF-β levels in the AML group and those in the control group (p < 0.01) was observed.
Conclusion IDO inhibition using novel IDO inhibitors along with chemotherapy is a promising approach to overcome the immune escape mechanisms in patients with AML, who exhibit increased levels of IDO expression and Tregs.

Keywords Acute myeloid leukemia, Flow cytometry, Indoleamine 2,3-dioxygenase, Real-time polymerase chain reaction, T regulatory cells

Article

RESEARCH

Blood Res 2024; 59():

Published online December 18, 2024 https://doi.org/10.1007/s44313-024-00048-0

Copyright © The Korean Society of Hematology.

Increased IDO expression and regulatory T cells in acute myeloid leukemia: implications for immune escape and therapeutic targeting

Raziyeh Hakak1, Behzad Poopak1,2* and Ahmad Majd1

1 Department of Cellular and Molecular Biology, Faculty of Biological Sciences, North Tehran Branch, Azad University, Tehran, Iran
2 Payvand Clinical and Specialty Laboratory, Tehran, Iran

Correspondence to:Behzad Poopak
bpoopak@gmail.com

Received: September 14, 2024; Accepted: December 4, 2024

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Abstract

Purpose This study aimed to determine the frequency of regulatory T cells (Tregs) (CD4+/FOXP3+) and indoleamine 2,3-dioxygenase (IDO) expression in patients with acute myeloid leukemia (AML).
Methods This cross-sectional case–control study was conducted between Jan 2022 and Dec 2023. Bone marrow samples were collected from 20 healthy individuals and 15 patients with AML. Flow cytometry, real-time polymerase chain reaction (PCR), and western blotting were used to evaluate the frequency of Treg and IDO expression levels.
Results The Treg percentage among total lymphocytes was lower in the AML group than that in the normal group. However, Treg percentage among T-helper (Th) lymphocytes was significantly higher in the AML group than that in the normal group (p < 0.05). The mean IDO expression in the AML group was significantly higher than that in the normal group (p = 0.004). A significant relationship was observed between IDO expression and Treg percentage among Th lymphocytes in the AML group (correlation = 0.637; p = 0.003). Moreover, western blot analysis showed a significant increase in IDO protein intensity in the AML group compared with that in the control group (p < 0.001). A significant difference was observed between the IDO concentrations in the AML group and that in the control group (p < 0.001). In addition, a significant difference between TGF-β levels in the AML group and those in the control group (p < 0.01) was observed.
Conclusion IDO inhibition using novel IDO inhibitors along with chemotherapy is a promising approach to overcome the immune escape mechanisms in patients with AML, who exhibit increased levels of IDO expression and Tregs.

Keywords: Acute myeloid leukemia, Flow cytometry, Indoleamine 2,3-dioxygenase, Real-time polymerase chain reaction, T regulatory cells

Fig 1.

Figure 1.Gating strategy (A) and frequency of CD4+ CD25+FoxP3.+ Tregs (B)

Fig 2.

Figure 2.a The Albumin qRT-PCR products analyzed using a 2% agarose gel. Results confirmed the expected unique 200 bp band for the Albumin gene, indicating specific amplification of Albumin. L: 100 bp ladder (Fermentas), 1& 2: analytical samples, NTC: no template control. b The IDO qRT-PCR products analyzed using a 2% agarose gel. The results confirmed the expected unique 122 bp band for the IDO gene, indicating specific IDO amplification. L: 100 bp ladder (Fermentas); 1, 2 & 3: analytical samples, NTC: no template control. c Comparison of mRNA levels of IDO between AML and control groups. (***p < 0.001)

Fig 3.

Figure 3.Analysis of IDO protein intensity in the AML and control groups using western blotting (**p < 0.01)

Fig 4.

Figure 4.Concentrations of IDO (a) and TGF-β (b) in the bone marrow of the AML and control groups. No significant correlation is observed between IDO and TGF-β levels (c) (**p < 0.01, ***p < 0.001)

Table 1 . Primers and reaction conditions used in qRT-PCR.

GeneSequenceProduct size (bp)
IDOF: 5′-TCC TGG ACA ATC AGT AAA GAG TAC C-3′122
R: 5′-TCA GGC AGA TGT TTA GCA ATG AAC -3′
AlbuminF: 5′-GCT ATC CGT GGT CCT GAA CC-3′200
R: 5′-CTT CTC AGA AAG TGT GCA TAT ATC TG-3′

Table 2 . Basic and clinical characteristics of patients in the two groups.

CharacteristicsNormal groupAML groupp-value
Gender
Male13 (65%)4 (7.26%)0.023
Female7 (35%)11 (3.73%)
Age, year33.27 ± 90.2833.24 ± 00.37≥ 0.001
Less than 5 years (toddler)4 (20%)2 (3.13%)
5–13 years (child)6 (30%)2 (3.13%)
14–19 years (adolescent)1 (5%)0 (0%)≥ 0.001
20–55 years (adult)3 (15%)7 (7.46%)
Over 55 years (older adult)6 (30%)4 (7.26%)
Subtype
Common B0 (0%)0 (0%)
M00 (0%)1 (7.6%)
M10 (0%)4 (7.26%)
M1/M20 (0%)1 (7.6%)
M20 (0%)1 (7.6%)
M4/M20 (0%)1 (7.6%)≥ 0.001
M50 (0%)2 (3.13%)
Non-M30 (0%)5 (3.33%)
Pro B0 (0%)0 (0%)
Relapsed B0 (0%)0 (0%)
Relapsed T0 (0%)0 (0%)
T0 (0%)0 (0%)
Blast percentage53.0 ± 87.099.28 ± 13.5≥ 0.001

Table 3 . Comparison of mean percentages of Tregs in patients in the two groups.

Treg percentageNormal groupAML groupp-value
Percentage of total Tregs00.0 ± 69.00.00 ± 55.00.309
Percentage of Tregs in total lymphocytes1.00 ±72.100.0 ± 55.560.023
Percentage of Tregs in T lymphocytes3.1 ± 32.994.1 ± 28.30.134
Percentage of Tregs in Th lymphocytes7.2 ± 25.979.2 ± 01.530.030

Table 4 . Correlation coefficients of IDO expressions with the percentages of Tregs.

Percentage TregNormal groupAML group
Correlation coefficientp-valueCorrelation coefficientp-value
Percentage of total Tregs083.00.729246.00.377
Percentage of Tregs in total lymphocytes350.00.130305.00.269
Percentage of Tregs in T lymphocytes251.00.286191.00.494
Percentage of Tregs in Th lymphocytes637.00.003164.00.559

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