Hematology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/yhem20

Correlation of the transcription factors IRF4 and

BACH2 with the abnormal NFATC1 expression in T
cells from chronic myeloid leukemia patients

Yikai Zhang, Xiangbo Zeng, Xianfeng Zha, Jing Lai, Guangxiao Tan, Shaohua
Chen, Xibao Yu, Yangqiu Li & Ling Xu

To cite this article: Yikai Zhang, Xiangbo Zeng, Xianfeng Zha, Jing Lai, Guangxiao Tan, Shaohua
Chen, Xibao Yu, Yangqiu Li & Ling Xu (2022) Correlation of the transcription factors IRF4 and
BACH2 with the abnormal NFATC1 expression in T cells from chronic myeloid leukemia patients,
Hematology, 27:1, 523-529, DOI: 10.1080/16078454.2022.2066245

To link to this article: https://doi.org/10.1080/16078454.2022.2066245

© 2022 The Author(s). Published by Informa View supplementary material

UK Limited, trading as Taylor & Francis
Group

Published online: 11 May 2022. Submit your article to this journal

Article views: 1006 View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=yhem20
HEMATOLOGY
2022, VOL. 27, NO. 1, 523–529
https://doi.org/10.1080/16078454.2022.2066245

Correlation of the transcription factors IRF4 and BACH2 with the abnormal
NFATC1 expression in T cells from chronic myeloid leukemia patients
Yikai Zhang a,b*, Xiangbo Zeng a*, Xianfeng Zha c
, Jing Lai a
, Guangxiao Tana, Shaohua Chen a
,
Xibao Yu a, Yangqiu Li a and Ling Xu a
a
Department of Hematology, First Affiliated Hospital, Institute of Hematology, School of Medicine, Key Laboratory for Regenerative
Medicine of Ministry of Education, Jinan University, Guangzhou, People’s Republic of China; bGuangzhou Municipality Tianhe Nuoya Bio-
engineering Co. Ltd, Guangzhou, People’s Republic of China; cDepartment of Clinical Laboratory, First Affiliated Hospital, Jinan University,
Guangzhou, People’s Republic of China

ABSTRACT KEYWORDS
Objective: T cell dysfunction is a common characteristic of patients with myeloid leukemia and CML; T cell dysfunction;
is closely related to clinical efficacy and prognosis. In order to clarify the mechanisms leading to NFATC1; IRF4; BACH2; T cell;
the T cell dysfunction, we characterized the gene expression profile of T cells from chronic immunotherapy
myelogenous leukemia (CML) patients by microarray analysis and investigated the related
regulating pathway.
Methods: We employed gene expression profiling, bioinformatics and real-time quantitative
reverse transcription PCR (RT-qPCR) to detect genes differentially expressed in CML patients
versus healthy donors.
Results: There were 1704 genes differentially expressed between CD3+ T cells from CML
patients and healthy donors, including 868 up-regulated genes and 836 down-regulated
genes, which mostly related to T cell functional pathways. In particular, lower expression of
NFATC1, a member of the TCR signaling pathway, was detected in CD3+ T cells from CML
patients. We further found that the expression of IRF4 and BACH2, transcription factors that
potentially regulate NFATC1, in CD3+ T cells from CML patients was significantly lower than
that in healthy donors.
Conclusion: We for the first time observed the altered gene expression profiles of CD3+ T cells
from CML patients, and the results suggested that IRF4, BACH2 and NFATC1 may be involved in
regulating T cell dysfunction in CML patients in the form of a transcriptional regulatory
network. These findings may provide potential targets for tyrosine kinase inhibitors in
combination with other targeted immunotherapies .

Introduction
Chronic myelogenous leukemia (CML) is a common
leukemia that is characterized by the BCR-ABL fusion
protein in adults [1]. Although tyrosine kinase inhibitor
(TKI) targeted therapy is beneficial for most CML
patients in the chronic phase, there is a portion of
patients who have primary TKI resistance or TKI-
induced ABL gene mutations as well as disease pro-
gression in the blast phase. Thus, new avenues for
improving this therapeutic strategy are needed [2–5].
Effective therapy for such CML patients includes hema-
topoietic stem cell transplantation (HSCT), including
allogenic-HSCT and haploidentical-HSCT; however, its
application is limited for older patients [6,7]. Thus,
exploration of novel therapeutic approaches such as
immunotherapy in combination with TKIs is necessary
for CML patients.
T cell immunity is an important component for
maintaining anti-tumor and antivirus capability in the
body; however, T cell immune deficiency is common
in patients with tumors and viral infections. In recent
years, anti-programmed death receptor (PD-1) [8]
and chimeric antigen receptor T (CAR-T) cell [9–15]
have significantly improved clinical outcome for lym-
phoma and leukemia patients in clinical trials. T cell
immunotherapy has become an important method
for tumor treatment. Our team and others have
found that T cell dysfunction is a common character-
istic of CML patients and that it is closely related to
disease status, prognosis, and TKI treatment effects
[6,16–19]. Based on abnormalities in molecules
related to the T cell receptor (TCR) signaling pathway
in T cells, such as CD3ζ and ZAP70, regulation of
these molecules could only partially restore T cell func-
tion [20–23]. Thus, further investigation of the mechan-
ism underlying T cell dysfunction is needed to
determine the regulatory approach and to enhance T
cells against leukemia.

CONTACT Yangqiu Li yangqiuli@hotmail.com; Ling Xu lingxu114@163.com

*These authors contributed equally to this work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/16078454.2022.2066245.
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrest-
ricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
524 Y. ZHANG ET AL.
Nuclear factor of activated T cells (NFAT), a critical tran-
scription factor of TCR signaling pathway, is involved in
the regulation of T cell activation, differentiation and dys-
function [24–27]. While we also found that expression of
NFATC1 was reduced in T cells from CML patients by gene
expression profiling in this study. However, the reason
for down-regulation of NFATC1 in T cells from CML
patients remains unclear. Therefore, in this study, we
focused on analyzing the differential gene expression
and related signaling pathway changes in T cells from
CML patients and healthy donors.
Materials and methods
Sample preparation
Peripheral blood from three newly diagnosed patients
with de novo CML (DN-CML) and four healthy individ-
uals (HIs) was collected for gene expression profiling
analysis. In order to verify the result of differentially
expressed genes, peripheral blood from 15 DN-CML
patients and 19 HIs were collected for RT-qPCR. Both
the gene expression profiling analysis and the RT-
qPCR used RNA as genetic material. There was no sig-
nificant difference in age between DN-CML patients
and healthy donors (p = 0.088). The diagnostic criteria
for DN-CML patients included in this study were in
accordance with the 2016 WHO diagnostic criteria
[28]. Samples were obtained in the Hematology
Department, First Affiliated Hospital of Jinan Univer-
sity. Ethical approval was obtained from the Ethics
Committee of the First Affiliated Hospital of Jinan Uni-
versity (20170302), and informed consent was pro-
vided by the patients and healthy donors. The
clinical information of CML patients and healthy
donors was provided in Supplementary Table 1.
CD3+ T cell sorting
Mononuclear cells were isolated from peripheral blood
by Ficoll-Hypaque gradient centrifugation. The enrich-
ment of CD3+ T cells was performed with CD3 MicroBe-
ads (130-050-101, Miltenyibiotec) as previously
described [29,30].
Gene expression profiling
CD3+ T cell RNA was used to perform global gene
expression profile analysis using the Affymetrix HG
U133 Plus 2.0 GeneChip (Shanghai Biochip Co., Ltd.,
Shanghai, China). An Affymetrix GeneChip® Scanner
3000 was used for raw data scanning.
RT-qPCR
Total RNA was prepared using TRIzol (15596018, Invi-
trogen, Theimo Fisher Scientific) reagent and then
used for cDNA synthesis using the High-Capacity
cDNA Reverse Transcription Kit (4368813, ABI,
Thermo Fisher Scientific) according to the manufac-
turer’s instructions. RT-qPCR analysis was performed
using SuperReal PreMix Plus (SYBR Green) (FP207
TIANGEN) according to the manufacturer’s protocol.
Each reaction was performed in a final volume of
20 μL containing 1 μL of the cDNA, 0.5 μL of each
primer, and 10 μL 2×SYBR Green PCR Master Mix.
The amplification profile was denaturation at 95°C
for 15 min, followed by 40 cycles of 95°C for 10 s,
60°C for 20 s, and 72°C for 20 s. At the end of the
RT-qPCR cycles, melting curve analyses were per-
formed as well as electrophoresis of the products
on 1.5% agarose gels in order to validate the
specific generation of the expected RT-qPCR
product [31]. ACTB was used as an internal control
for NFATC1, NFATC2, NFATC3, IRF4 and BACH2. ΔCt
was employed to evaluate the gene expression. The
oligonucleotide sequences are provided in Sup-
plementary Table 2.
Statistical analysis
R (version 4.0.4) was used for statistical analysis and
graphics in this study. Raw data were read and normal-
ized by the RMA method in the ‘Affy’ package. Differ-
ential gene expression analysis was performed with
the ‘limma’ R package. Genes with a p-value less than
0.05 and fold change greater than 1.5 or less than
−1.5 were regarded differentially expressed and
further examined by Kyoto Encyclopedia of Genes
and Genomes (KEGG) and Gene Ontology (GO) enrich-
ment analysis using the ‘clusterProfiler’ package. The
packages ‘GOplot’, ‘ggplot2’, and ‘pheatmap’ were
used to plot images. Protein–Protein Interaction (PPI)
analysis was performed with the ‘STRINGdb’ R
package. Packages ‘Affy’, ‘clusterProfiler’, ‘limma’ and
‘STRINGdb’ were downloaded from Bioconductor
(http://bioconductor.org/). Packages ‘GOplot’,
‘ggolot2’ and ‘pheatmap’ were downloaded from
CRAN (https://cran.r-project.org/). Comparison of the
gene expression levels in the DN-CML and HI groups
was performed by the Mann–Whitney U test, and p <
0.05 was regarded as statistically significant. The
potential transcriptional regulators of NFATC1 were
predicted using hTFtarget database (http://bioinfo.
life.hust.edu.cn/hTFtarget/#!/).
Results
Significantly altered expression of functionally
related genes in T cells from CML patients
To further clarify the potential regulatory mechanisms
leading to specific T cell clonal dysfunction, we col-
lected peripheral blood CD3+ T cells from three
 HEMATOLOGY 525

chronic CML patients and four healthy donors for
microarray analysis. There were 1704 differentially
expressed genes (DEGs) when comparing CD3+ T
cells from CML patients with healthy donor T cells,
including 868 up-regulated genes and 836 down-regu-
lated genes (Figure 1(A, B)). We then performed KEGG
signaling (Figure 1(C)) and biological process (GO-BP)
analysis (Figure 1(D)) for enriched pathways using
the DEGs. We found that several immune-related
KEGG pathways are enriched in CML CD3+ T cells
(marked by asterisks), including the T cell receptor sig-
naling pathway. In addition, there were seven GO-BPs
related to T cell function that were altered, including T
cell activation, proliferation, differentiation, co-stimu-
lation, apoptosis, T cell homeostasis, and T cell acti-
vated regulation.
Down-regulated NFATC1 expression in the TCR
signaling pathway in CD3+ T cells from CML
patients
A total of 68 DEGs which were involved in regulating
these T cell biological pathways were revealed
(Figure 2(A)). In this study, we mainly focused on the
genes enriched in the TCR signaling pathway which
is one of the important signaling pathways in regulat-
ing T cell function (Figure 2(B)). There were 20 DEGs
involved in regulation of the TCR signaling pathway,
and these included NFATC1 (p < 0.05, FC = 1.644), a
key transcription factor relatively close to downstream
of the TCR signaling pathway, which was down-
regulated.
Decreased expression of IRF4- and BACH2-
mediated regulation of NFATC1 in CD3+ T cells
from CML patients
To determine potential reasons for NFATC1 down-regu-
lation in T cells from CML patients. We compared the
results which predicted by hTFtarget database with
the DEGs identified by microarray analysis. As shown
in Figure 3(A,B), there were 24 potential transcriptional
regulators of NFATC1 that were altered. We next ana-
lyzed the correlation between the 24 transcription
factors and NFATC1 (Figure S1). The results demon-
strated that 21 transcription factors correlated with
NFATC1, including eight genes that were positively cor-
related and thirteen genes that were negatively corre-
lated. It has been reported that BTB domain and CNC
homolog 2 (BACH2) [32–34] and interferon regulating
factor 4 (IRF4) [35,36] are involved in regulating T cell
function. It has been reported that NFAT family
members NFATC1, NFATC2, and NFATC3 are also
expressed in T cells and participate in the regulation
of T cell function [37–40]. Thus, we examined the
expression level of BACH2, IRF4, NFATC1, NFATC2, and
NFATC3 in the CD3+ T cells from CML patients and
healthy donors. As shown in Figure 3(C), the expression
of BACH2 (p < 0.01), IRF4 (p < 0.01), NFATC1 (p < 0.01),
and NFATC3 (p < 0.001) in CML patient T cells was sig-
nificantly lower than that in healthy donors. We

Figure 1. Gene differentially expressed in T cells from CML patients and healthy donors. (A) The different gene expression profiles.
Differentially expressed genes were selected based on a FC >1.5 or a FC <−1.5 with a p < 0.05. Red: Up-regulated genes; Blue:
Down-regulated genes; Gray: other genes. The ‘limma’ R package was used for analysis. (B) Heatmap of differentially expressed
genes between CML patients and healthy donors. (C) KEGG signaling pathway enrichment analysis. White asterisks: Signaling
pathways related to T cells. The ‘clusterProfiler’ R package was used for analysis. (D) GO enrichment analysis. The ‘clusterProfiler’
R package was used for analysis. The R (version 4.0.4) was used for analysis and graphics in this figure.
526 Y. ZHANG ET AL.

Figure 2. The different genes associated with T cell function in CD3+ T cells from CML patients and healthy donors. (A) The
relationship between the different genes and T cell biological behaviors. The ‘GOplot’ R package was used for graphics. (B)
Heatmap of different genes related to the TCR signaling pathway. The R (version 4.0.4) was used for graphics in this figure.

further analyzed the correlation between these genes were positively correlated with NFATC1 (Figure 3(D)).
and NFATC1, and the results demonstrated that the The PPI analysis also shown that there was an inter-
expression of IRF4 (R = 0.538, p = 0.047), BACH2 (R = action between NFATC1 and IRF4, while BACH2 shown
0.697, p = 0.006) and NFATC3 (R = 0.875, p < 0.001) an interaction with IRF4 (Figure 3(E)).

Figure 3. Expression of transcription factors that potentially regulate NFATC1 in CD3+ T cells from CML patients and healthy
donors. (A) The expression of 24 transcription factors that may regulate NFATC1 in CML patients and healthy donors. (B) Left:
Screening strategy for the 24 potential transcription factors; Right: The predicted genomic positions of the BACH2 and IRF4
that regulate NFATC1. (C) RT-qPCR results for BACH2, IRF4, NFATC1, NFATC2, NFATC3. Mean of two normally distributed variables
was compared by independent samples Student’s test. The results are graphing the standard deviation. ** p < 0.01, **** p < 0.001.
(D) Correlations between IRF4, NFATC3, BACH2 and NFATC1 from RT-qPCR results. The Pearson’s Correlation was used to analyze
the linear relationship between each gene. (E) The interaction of NFATC1, IRF4 and BACH2 that is analyzed by PPI analysis. The R
(version 4.0.4) was used for analysis and graphics in this figure.
 HEMATOLOGY 527

Discussion subfamily 3 group C member 1), USF1 (upstream tran-

scription factor 1), MNT (MAX network transcription
As important members of the immune system, T cells
repressor), BACH2, IRF4, NCOR1 (nuclear receptor core-
mainly play the role of scavenging and killing infected
pressor 1), and SIN3A (SIN3 transcription regulator
target or tumor cells. Thus, whether the function of T
family member A) i.e. transcription factors that may
cells is sound is closely related to the immune status
potentially regulate NFATC1, was decreased in CML
and prognosis of tumor patients. In our previous
patient T cells. Among these transcription factors,
studies, we found that there were specific anti-leuke-
IRF4 [35,36] and BACH2 [32–34]. have been widely
mia T cell clones in myeloid leukemia patients, but
studied and are closely related to T cell function.
these clones were defective in activation, differen-
The IRF transcription factor family consists of nine
tiation, proliferation, and immune function; thus,
members, IRF1 to IRF9. This family plays an important
these clones are unable to exert anti-leukemia effects
role in regulating innate and adaptive immune
[16,18,20–23,41]. The TCR signaling pathway is the
responses and tumorigenesis. IRF4 is closely related
first signaling pathways in T cells that exerts immune
to IRF8 and was initially identified as a nuclear factor
functions. Previous studies from our group have
[50]. This protein plays an important role in the differ-
shown that CD3ζ and ZAP70, which are important mol-
entiation and functional regulation of CD4+ and CD8+
ecules in the TCR signaling pathway, are down-regu-
T cells [51]. In mice, Irf4 is necessary for the protective
lated in CD3+ T cells in CML patients [21,42,43].
effects of CD8+ T cells during infection [35]. A recent
However, the mechanism underlying T cell dysfunction
study has further shown that IRF4 is an important regu-
in myeloid leukemia patients is still not clear. To under-
latory factor in the formation of CD8+ memory T cells,
stand the mechanism of T cell dysfunction in this
and it is necessary for the effective reactivation of CD8+
study, we performed gene expression profiling analysis
T cells [36]. Moreover, the results predicted by hTFtar-
and found that the expression of NFATC1, which is a
get database shown that IRF4 may regulate the
key transcription factor in the TCR signaling pathway,
expression of NFATC1, while our recent results have
was significantly reduced in CD3+ T cells from CML
also demonstrated that NFATC1 is necessary for
patients. The NFAT family consists of five members:
human CD8+ T cells to perform their normal immune
NFATC1 (also known as NFATc or NFAT2), NFATC2
function [30]. These results suggested that T cell dys-
(also known as NFATp or NFAT1), NFATC3 (also known
function in CML patients may be associated with the
as NFATx or NFAT4), NFATC4 (also known as NFAT3),
reduced expression of IRF4 and NFATC1.
and NFAT5 (also known as TonEBP or OREBP). T cells
In addition, we found that BACH2, another transcrip-
mainly express NFATC1, NFATC2, and NFATC3, and
tion factor that potentially regulates NFATC1, was
these molecules are important transcription factors
decreased in CML patient T cells. Although the PPI
involved in the regulation of T cell function [38].
analysis result showed that there was no direct inter-
NFATC1 is an important member of the NFAT family,
action between BACH2 and NFATC1, the RT-qPCR
and its classical signaling involves TCR and co-stimu-
results in this study showed that BACH2 expression
latory signal activation. Research in mice has shown
was positively correlated with NFATC1. One study has
that NFATC1/2 deletion causes a decrease in T cell cyto-
shown that up-regulating BACH2 can inhibit the
kine secretion [37], T cell dysfunction, exhaustion, and
program of terminal exhaustion molecules via tran-
self-tolerance [26,44]. Recently, it has been found that
scriptional repression and epigenetic silencing [34].
NFATC1 and NFATC2 are involved in regulating the for-
These data suggested that down-regulation of BACH2
mation of the memory and effector CD8+ T cell subsets
may be also related to T cell dysfunction in CML
in mice [45,46]. Earlier studies have found that NFATC1,
patients. Interestingly, our previous results have
NFATC2, and NFATC3 regulate the expression of IL-4
shown that the distribution of memory T cells in CML
and IFN-γ in mouse CD4+ T cells, thus affecting the
patients was skewed from naïve and central memory
differentiation of Th1 and Th2 cells [38,47–49]. These
T cells to effector memory and effector T cells [52], in
studies demonstrated the important role of the NFAT
addition, we also found that there was an increased
family in regulating the activation, differentiation,
percentage of CD8+ T cell subset among the CD3+ T
and immune function of mouse T cells. Recently, we
cell population in CML patients (Unpublished data).
also demonstrated that NFATC1 is necessary for
All of these alternations seem that the adaptive
normal human CD8+ T cell immune function [30].
immune reaction was activated in CML patients,
Based on the above, down-regulation of NFATC1 may
however, the current finding suggested that in the
be related to the dysfunction of CD3+ T cells in CML
level of genetic, key transcription factors which
patients.
response for activation, protective function, and inhi-
Therefore, we further explored potential reasons for
bition of exhaustion of T cells were down-regulated.
down-regulation of NFATC1 in CML patient T cells and
Further research needs to understand the clear mol-
found that the expression of SMC1A (structural main-
ecular mechanisms behind these changes more
tenance of chromosomes 1 A), NR3C1 (nuclear receptor
deeply.
528 Y. ZHANG ET AL.
Conclusion
In summary, we found that the expression of IRF4 and
BACH2, potential transcription factors that regulate
NFATC1, are significantly down-regulated in CML
patient T cells, and the expression of IRF4 and BACH2
are positively correlated with NFATC1. These prelimi-
nary results suggest that decreased expression of
IRF4 and BACH2 may be potential factors leading to
the down-regulation of NFATC1 in CML patient T
cells. These findings will deepen our understanding
of T cell dysfunction in CML patients and provide
potential targets for CML immunotherapy.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This study was supported by grants from the National
Natural Science Foundation of China [Nos. 81770152,
82070152, and 82000108], Guangdong Basic and Applied
Basic Research Foundation [No. 2021A1515110004], and
China Postdoctoral Science Foundation [No. 2021M701427].
Data availability statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
ORCID
Yikai Zhang http://orcid.org/0000-0002-6851-6260
Xiangbo Zeng http://orcid.org/0000-0002-3176-8800
Xianfeng Zha http://orcid.org/0000-0002-4970-1305
Jing Lai http://orcid.org/0000-0002-9581-7325
Shaohua Chen http://orcid.org/0000-0003-4945-5914
Xibao Yu http://orcid.org/0000-0003-0643-8372
Yangqiu Li http://orcid.org/0000-0002-0974-4036
Ling Xu http://orcid.org/0000-0002-7044-7663
References
[1] Chopra R, Pu QQ, Elefanty AG. Biology of BCR-ABL.
Blood Rev. 1999;13(4):211–229.
[2] Druker BJ. Activity of a specific inhibitor of the BCR-ABL
tyrosine kinase in the blast crisis of chronic myeloid leu-
kemia and acute lymphoblastic leukemia with the
Philadelphia chromosome. N Engl J Med. 2001;344
(14):1038–1042.
[3] Marin D, Milojkovic D, Olavarria E, et al. European
LeukemiaNet criteria for failure or suboptimal response
reliably identify patients with CML in early chronic
phase treated with imatinib whose eventual outcome
is poor. Blood. 2008;112(12):4437–4444.
[4] Jangamreddy JR, Panigrahi S, Lotfi K, et al. Mapping of
apoptin-interaction with BCR-ABL1, and development
of apoptin-based targeted therapy. Oncotarget.
2014;5(16):7198–7211.
[5] Liu Z, Shi Y, Yan Z, et al. Impact of anemia on the out-
comes of chronic phase chronic myeloid leukemia in
TKI era. Hematology. 2020;25(1):181–185.
[6] Hehlmann R, Hochhous A, Baccarani M, et al.
Chronic myeloid leukaemia. Lancet. 2007;370
(9584):342–350.
[7] Xu L, Chen H, Chen J, et al. The consensus on indi-
cations, conditioning regimen, and donor selection of
allogeneic hematopoietic cell transplantation for hem-
atological diseases in China-recommendations from
the Chinese Society of Hematology. J Hematol Oncol.
2018;11(1):33.
[8] Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade
induces responses by inhibiting adaptive immune
resistance. Nature. 2014;515(7528):568–571.
[9] Brudno JN, Somerville RPT, Shi V, et al. Allogeneic T cells
that express an anti-CD19 chimeric antigen receptor
induce remissions of B-cell malignancies that progress
after allogeneic hematopoietic stem-cell transplan-
tation without causing graft-versus-host disease. J Clin
Oncol. 2016;34(10):1112–1121.
[10] Wang H, Kaur G, Sankin AI, et al. Immune checkpoint
blockade and CAR-T cell therapy in hematologic malig-
nancies. J Hematol Oncol. 2019;12:59.
[11] Zhao JJ, Song YP, Liu DL. Clinical trials of dual-target
CAR T cells, donor-derived CAR T cells, and universal
CAR T cells for acute lymphoid leukemia. J Hematol
Oncol. 2019;12:17.
[12] Zhao Q. Novel chimeric antigen receptor T cells based
on T-cell receptor-like antibodies. Blood Sci. 2019;1
(2):144–147.
[13] Zhao R, Cui Y, Li S, et al. Current status and hurdles for
CAR-T cell immune therapy. Blood Sci. 2019;1(2):148–
155.
[14] Taefehshokr N, Baradaran B, Baghbanzadeh A, et al.
Promising approaches in cancer immunotherapy.
Immunobiology. 2020;225(2):151875.
[15] Zhang H, Zhao P, Huang H. Engineering better chimeric
antigen receptor T cells. Exp Hematol Oncol. 2020;9
(1):34.
[16] Li YQ, Yang LJ, Chen SH, et al. T cell receptor V beta
repertoire usage and clonal expansion of T cells in
chronic myelogenous leukemia. Chin Med J. 2004;117
(6):840–843.
[17] Rohon P, Porkka K, Mustjoki S. Immunoprofiling of
patients with chronic myeloid leukemia at diagnosis
and during tyrosine kinase inhibitor therapy. Eur J
Haematol. 2010;85(5):387–398.
[18] Zha X, Chen S, Yang L, et al. Characterization of the
CDR3 structure of the Vbeta21T cell clone in patients
with P210(BCR-ABL)-positive chronic myeloid leukemia
and B-cell acute lymphoblastic leukemia. Hum
Immunol. 2011;72(10):798–804.
[19] Yao DL, Xu L, Tan JX, et al. Re-balance of memory T cell
subsets in peripheral blood from patients with CML
after TKI treatment. Oncotarget. 2017;8(47):81852–
81859.
[20] Li YQ, Geng SX, Yin QS, et al. Decreased level of recent
thymic emigrants in CD4+ and CD8+ T cells from CML
patients. J Transl Med. 2010;8:47.
[21] Chen SH, Zha XF, Shi L, et al. Upregulated TCR zeta
improves cytokine secretion in T cells from patients
with AML. J Hematol Oncol. 2015;8:72.
[22] Xu L, Lu YH, Lai J, et al. Characteristics of the TCR V beta
repertoire in imatinib-resistant chronic myeloid leuke-
mia patients with ABL mutations. Sci China-Life Sci.
2015;58(12):1276–1281.

[23] Zha XF, Xu L, Chen SH, et al. Generation of V alpha 13/
beta 21(+)T cell specific target CML cells by TCR gene
transfer. Oncotarget. 2016;7(51):84246–84257.
[24] Gao R, Zhang Y, Zeng C, et al. The role of NFAT in the
pathogenesis and targeted therapy of hematological
malignancies. Eur J Pharmacol. 2022;921:174889.
[25] Hogan PG. Calcium-NFAT transcriptional signalling in T
cell activation and T cell exhaustion. Cell Calcium.
2017;63:66–69.
[26] Schietinger A, Greenberg PD. Tolerance and exhaus-
tion: defining mechanisms of T cell dysfunction.
Trends Immunol. 2014;35(2):51–60.
[27] Martinez GJ, Pereira RM, Äijö T, et al. The transcription
factor NFAT promotes exhaustion of activated CD8+
T cells. Immunity. 2015;42(2):265–278.
[28] Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision
to the World Health Organization classification of
myeloid neoplasms and acute leukemia. Blood.
2016;127(20):2391–2405.
[29] Zhang Y, Xu L, Chen S, et al. Identification of TCR V11-2-
D1-J1-1T cell clone specific for WT1 peptides using
high-throughput TCR gene sequencing. Biomark Res.
2019;7:12.
[30] Zhang Y, Wu J, Zeng C, et al. The role of NFAT2/miR-
20a-5p signaling pathway in the regulation of CD8
naïve T cells activation and differentiation.
Immunobiology. 2021;226(4):152111.
[31] Wu Y, Hu Y, Yu X, et al. TAL1 mediates imatinib-induced
CML cell apoptosis via the PTEN/PI3 K/AKT pathway.
Biochem Biophys Res Commun. 2019;519(2):234–239.
[32] Tsukumo S-i, Unno M, Muto A, et al. Bach2 maintains T
cells in a naive state by suppressing effector memory-
related genes. Proc Natl Acad Sci U S A. 2013;110
(26):10735–10740.
[33] Roychoudhuri R, Clever D, Li P, et al. BACH2 regulates
CD8(+) T cell differentiation by controlling access of AP-
1 factors to enhancers. Nat Immunol. 2016;17(7):851–860.
[34] Yao C, Lou G, Sun H-W, et al. BACH2 enforces the tran-
scriptional and epigenetic programs of stem-like CD8T
cells. Nat Immunol. 2021;22(3):370–380.
[35] Raczkowski F, Ritter J, Heesch K, et al. The transcription
factor interferon regulatory factor 4 is required for the
generation of protective effector CD8+ T cells. Proc
Natl Acad Sci U S A. 2013;110(37):15019–15024.
[36] Harberts A, Schmidt C, Schmid J, et al. Interferon regu-
latory factor 4 controls effector functions of CD8
memory T cells. Proc Natl Acad Sci U S A. 2021;118
(16):e2014553118.
[37] Peng SL, Gerth AJ, Ranger AM, et al. NFATc1 and
NFATc2 together control both T and B cell activation
and differentiation. Immunity. 2001;14(1):13–20.
HEMATOLOGY 529
[38] Macian F. NFAT proteins: key regulators of T-cell devel-
opment and function. Nat Rev Immunol. 2005;5(6):472–
484.
[39] Koch S, Reppert S, Finotto S. NFATc1 deletion in T
lymphocytes inhibits the allergic trait in a murine
model of asthma. Clin Exp Allergy. 2015;45(8):1356–
1366.
[40] Klein-Hessling S, Rudolf R, Muhammad K, et al. A
threshold level of NFATc1 activity facilitates thymocyte
differentiation and opposes notch-driven leukaemia
development. Nat Commun. 2016;7:11841.
[41] Li Y, Geng S, Du X, et al. Restricted TRBV repertoire in
CD4 + and CD8+ T-cell subsets from CML patients.
Hematology. 2011;16(1):43–49.
[42] Zha X, Chen S, Yang L, et al. Upregulated TCRζ enhances
interleukin-2 production in T-cells from patients with
CML. DNA Cell Biol. 2012;31(11):1628–1635.
[43] Zha X, Yan X, Shen Q, et al. Alternative expression of
TCRζ related genes in patients with chronic myeloid
leukemia. J Hematol Oncol. 2012;5:74.
[44] Martinez GJ, Pereira RM, Aijo T, et al. The transcription
factor NFAT promotes exhaustion of activated CD8(+)
T cells. Immunity. 2015;42(2):265–278.
[45] Klein-Hessling S, Muhammad K, Klein M, et al. NFATc1
controls the cytotoxicity of CD8(+) T cells. Nat
Commun. 2017;8:511.
[46] Xu TH, Keller A, Martinez GJ. NFAT1 and NFAT2 differen-
tially regulate CTL differentiation upon acute viral infec-
tion. Front Immunol. 2019;10:184.
[47] Kiani A, Viola JP, Lichtman AH, et al. Down-regulation of
IL-4 gene transcription and control of Th2 cell differen-
tiation by a mechanism involving NFAT1. Immunity.
1997;7(6):849–860.
[48] Porter CM, Clipstone NA. Sustained NFAT signaling pro-
motes a Th1-like pattern of gene expression in primary
murine CD4(+) T cells. J Immunol. 2002;168(10):4936–
4945.
[49] Rengarajan J, Tang B, Glimcher LH. NFATc2 and NFATc3
regulate T(H)2 differentiation and modulate TCR-
responsiveness of naive T-H cells. Nat Immunol.
2002;3(1):48–54.
[50] Huber M, Lohoff M. IRF4 at the crossroads of effector T-
cell fate decision. Eur J Immunol. 2014;44(7):1886–
1895.
[51] Nam S, Lim JS. Essential role of interferon regulatory
factor 4 (IRF4) in immune cell development. Arch
Pharm Res. 2016;39(11):1548–1555.
[52] Yao D, Xu L, Tan J, et al. Re-balance of memory T cell
subsets in peripheral blood from patients with CML
after TKI treatment. Oncotarget. 2017;8(47):81852–
81859.