Acute myeloid leukemia (AML) is the most common acute leukemia in adults with around 20,000 new diagnoses each year in the USA. The mainstream of treatment for AML is induction chemotherapy followed by post-remission consolidation. Allogeneic hematopoietic stem cell transplantation in many clinical settings can significantly improve survival. However, the overall prognosis remains poor with a 5-year survival rate of only 25%. Novel effective leukemia therapeutics is urgently needed.
Modulating the immune response to improve anti-tumor immunity provides a promising strategy for cancer treatment [
]. Studies using reagents inhibiting negative immune regulatory pathways, such as programmed cell death protein 1 (PD-1), have achieved great success [
]. This strategy targets T cell exhaustion, a status of T cell dysfunction that contributes to compromised anti-tumor T cell responses. Exhausted T cells gradually lose their capacity for cytokine production and cell killing, eventually undergo apoptosis and deletion [
]. Upregulation of PD-1 and other inhibitory pathways such as T cell immunoglobulin domain and mucin domain 3 (TIM-3), 2B4, Lymphocyte-activation gene 3 (LAG-3), and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is not only a hallmark, but also an important mechanism involved in the development of T cell exhaustion [
]. Studying the role of inhibitory pathways in AML is appealing. Several reports including ours have shown that inhibitory receptors including PD-1, TIM-3, and TIGIT are elevated on T cells and associate with immune suppression in AML [
]. Combined blockade of PD-1 and TIM-3 pathways synergistically reduced tumor burden and leukemia death in a mouse model of AML [
]. These data suggest that targeting the inhibitory pathways to restore T cell function and anti-tumor immune response may represent an effective leukemia therapy. Moving this strategy forward to clinical applications is under active development, but it is also important to understand the transcriptional mechanisms involved in the regulation of these inhibitory receptors in AML, which is currently unknown.
B lymphocyte-induced maturation protein 1 (Blimp-1) is a zinc finger-containing transcription factor functioning as a decision maker for memory B cell differentiation [
]. Recent studies in mouse models of infection uncovered its crucial role in regulating CD8
T cell exhaustion [
]. Here, we examine the effect of Blimp-1 in the pathogenesis of AML using blood samples collected from a cohort (
= 24) of patients with AML at initial diagnosis. We demonstrate an inhibitory role for Blimp-1 on T cell response in AML. Elevated expression of Blimp-1 in T cells associates with upregulation of inhibitory receptors and reduced T cell capacity of cytokine production and cytotoxicity, features which are consistent with exhaustion. Importantly, Blimp-1 knockdown by siRNA reverses these functional defects. In addition, Blimp-1 binds to the promoters of PD-1 and TIGIT and upregulates their expression, suggesting that the suppressive effect of Blimp-1 on the T cell response is mediated by its transcriptional regulation of PD-1 and TIGIT. Our studies demonstrate that Blimp-1 is an important regulator of T cell exhaustion in AML and thus an attractive target for effective leukemia therapeutics.
Peripheral blood collected from AML patients were obtained from the tissue bank maintained by the Penn State Hershey Cancer Institute of Penn State University College of Medicine, Hershey, PA. The study was approved by the Institutional Review Board of Penn State University College of Medicine. Full informed consent was obtained from all patients. Samples from 24 patients (10 males and 14 females, age 57 ± 15 years, range, 23–77 years) diagnosed with AML per WHO classification were used in the study. Samples of 25 healthy volunteers (13 males and 12 females, age 55 ± 15 years, range, 21–77 years) were obtained as controls.
Immunofluorescence staining and flow cytometric analysis
For surface staining, cells were incubated at room temperature with human Fc block (BD Biosciences, San Diego, CA, USA) and followed by staining with directly conjugated mAbs for 30 min at 4 °C. The mAbs used were anti-human CD3-BV605, CD4-V500, CD8-APC-H7, CD45RA-BV421, CCR7-PerCp-Cy5.5, PD-1-PE-Cy7, CD160-Alexa Fluor 488 (BD Biosciences), CD4-FITC, TIM-3-BV421, 2B4-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA), and TIGIT-APC (eBioscience, San Diego, CA, USA). Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences). FlowJo Software (Tree Star, Ashland, OR, USA) was used in data analysis.
Lyophilized SmartFlare probe (Merck Millipore, Guyancourt, France) was used to detect Blimp-1 mRNA following the manufacturer’s instruction.
In vitro stimulation and intracellular staining
PBMCs were stimulated with anti-CD3/CD28 (2 and 5 μg/mL), plus Golgiplug (BD Pharmingen) for 5 h before intracellular staining Blimp-1-PE, IFN-γ-PE-CF594, and IL-2-PerCp-Cy5.5 (BD Pharmingen). For perforin study, perforin-PE-CF594 (BD Pharmingen) was used. A Fixable Viability Dye eFluor 450 (eBioscience) was used to assess cell viability.
SMARTpool of siRNA for Blimp-1 and control were obtained from GE Dharmacon RNA Technologies (GE Dharmacon, Lafayette, CO, USA). Control and specific siRNAs were added at a final concentration of 1 μM per well for 72 h. For functional assays, cells were further stimulated with anti-CD3/CD28 for 5 h, followed by flow analysis.
Plasmid construction, transfection, and real-time PCR
plasmid (RGS-6xHis-BLIMP-1-pcDNA3.1-) was a gift from Adam Antebi [
cDNA was cloned into pcDNA3.1+ plasmid. The
gene promoter (−1063/+70 bp relative to the transcription start site) and
promoter (−2228/+76 bp) were cloned into pGL3-basic.
plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Specific transcripts were quantified by real-time PCR with TaqMan probes according to the manufacturer’s instructions (Thermo Fisher Scientific).
Luciferase reporter assay
293T cells were transfected with a mixture of the indicated expression plasmids. After 24 h, luciferase assays were performed using a dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were conducted as previously described [
]. Briefly, T cells were stimulated in vitro with anti-CD3 [
] for 48 h followed by cross-linking, sonication, and chromatin immunoprecipitation with antibodies to Blimp-1 or normal goat IgG (Abcam, Cambridge, UK). DNA was then quantified by real-time PCR. Primer sequences were provided in Additional file
: Supplemental data.
GraphPad5 (GraphPad Software, La Jolla,CA, USA) was used for statistical calculations. The normality of each continuous variable was evaluated using the Kolmogorov–Smirnov test. For data distributed normally, the comparison of variables was performed using unpaired or paired (where specified) Student’s
t test. For data not distributed normally, the comparison of variables was performed with a Mann–Whitney
U test or a Wilcoxon signed-rank test for unpaired and paired data, respectively. Comparisons of categorical patient characteristics were analyzed using Fisher’s exact test. To evaluate correlation, Pearson’s correlation coefficients were used. All tests are two-tailed with
P values less than 0.05 considered statistically significant.
Blimp-1, encoded by the
gene, was initially identified as a transcriptional repressor regulating terminal differentiation of B cells into plasma cells [
]. The effect of Blimp-1 in lymphoproliferative disorders has been well studied [
]. Recent studies using mouse models of virus infection elucidated its role in T cell differentiation. During acute viral infections, Blimp-1 promotes the differentiation of CD8
T cells into short-lived terminal effectors while dampening the formation of long-lived central memory T cells [
]. During chronic viral infection, Blimp-1 enhances expression of inhibitory receptors and promotes development of T cell exhaustion [
]. Notably, haploinsufficient mice which had intermediate expression of Blimp-1 controlled chronic virus infection better than either wild type or Blimp-1fully deficient mice, indicating that a moderate amount of Blimp-1 facilitates effector mechanisms without causing T cell exhaustion [
]. These findings suggest a complex role for Blimp-1 in regulating T cell response. Although well studied in viral infection, the T cell regulatory role of Blimp-1 in tumor immunity has not been fully defined and the effect of Blimp-1 on anti-leukemia response is unknown. In this study, phenotypic and functional analyses of PBMCs collected from AML patients were performed. We focused on dissecting the role of Blimp-1 in modulating the T cell response in AML. Our study demonstrates that expression of Blimp-1 in both CD4
T cells is significantly increased in AML patients compared to that in healthy donors. Consistent with exhaustion, Blimp-1
T cells express high levels of co-inhibitory receptors such as PD-1 and TIGIT. In addition, they are phenotypically skewed toward terminal effector differentiation and functionally impaired in their production of cytokines and potential for cytotoxicity. Importantly, the functional defect is reversed by inhibition of Blimp-1 via siRNA knockdown. To our knowledge, this study is the first to display an immune suppressive role of Blimp-1 in AML. Our finding suggests that Blimp-1 associates with T cell exhaustion and suppresses T cell function, which may subsequently impair anti-leukemia immune response. Therefore, targeting Blimp-1 may provide effective therapeutics for AML.
We observed a wide variation of Blimp-1 expression in T cells among AML patients. Clinically, the initial presentation of AML is highly heterogeneous [
]. Some patients seek medical attention earlier during the disease course due to their high sensitivity to leukemia-related symptoms or occasionally incidental abnormal laboratory findings; others present later when the leukemia has developed for a longer period of time. The large variation of Blimp-1 expression among the AML patients may represent their different disease status. In fact, we found a significant association of Blimp-1 expression with the number of circulating leukemia blast. Patients who express high levels of Blimp-1 in their CD4
T cells present with high blast counts, indicating a correlation of Blimp-1 expression to late phase leukemia development. This situation might provide persistent leukemia antigen that is ideal for induction of T cell exhaustion, which is consistent with our finding that Blimp-1
T cells associate with exhaustion and display functional impairment. Thus, we speculate that treatment approaches targeting T cell exhaustion may be more effective in patients with higher expression of Blimp-1 as T cells in this patient population are more likely exhausted. Therefore, testing Blimp-1 expression in T cells might provide a crucial biomarker for effective leukemia treatment. Although promising, further studies of large size of samples are needed to make a definitive conclusion.
The mechanisms by which Blimp-1 regulates T cell responses are not fully understood. In our study, we observed a strong correlation between Blimp-1 expression and upregulation of inhibitory receptors such as PD-1 and TIGIT. Several studies have demonstrated an important role of PD-1 in inhibiting anti-leukemia T cell responses [
]. In addition, our recent study revealed that TIGIT contributes to T cell impairment in AML and associates with poor clinical outcomes [
]. We hypothesize that in AML, Blimp-1 suppresses T cell function through positive regulation of these inhibitory pathways. In the present study, we demonstrated a strong binding of Blimp-1 protein to the promoters of the genes encoding PD-1 and TIGIT. Importantly, inhibition of Blimp-1 by siRNA knockdown significantly decreased mRNA expression of PD-1 and TIGIT in T cells collected from AML patients. Consistently, cells overexpressing Blimp-1 showed upregulation of PD-1 and TIGIT. Therefore, Blimp-1 is a transcriptional regulator for these two important inhibitory receptors. This likely contributes to the mechanisms by which Blimp-1 suppresses T cell function in AML. An equally important question is how and why Blimp-1 is upregulated in AML. In viral infection, Blimp-1 expression is induced during T cell activation upon viral antigen stimulation [
]. Cytokines including IL-2 have been reported to be crucial mediators for the upregulation of Blimp-1. In AML, it has been observed that serum level of IL-2 is increased in AML patients, and the level is particularly higher in patients with high WBC at initial presentation [
]. Consistently, we observed a positive correlation between the high level of WBC and Blimp-1 expression in our study. We speculate that IL-2 and/or other cytokines may contribute to the regulation of Blimp-1 in AML. Further studies are warranted to address this important question.
In contrast to our finding that Blimp-1 upregulates the expression of PD-1, Lu et al. have reported that Blimp-1 inhibits CD8
T cell expression of PD-1[
]. Of note, their conclusion was drawn from a study of acute viral infection, in which PD-1 was increased shortly (hours) after antigen stimulation. The regulation mechanisms may be significantly different in the setting of chronic infections or cancer. Consistent with our finding, it has been reported that PD-1
T cells expressed a high level of Blimp-1 in patients with chronic lymphocytic leukemia [
]. In addition, studies using mouse models of viral infection have demonstrated that Blimp-1 enhanced the expression of inhibitory receptors on exhausted T cells during chronic viral infection and conditional deletion of Blimp-1 reversed this process [
]. Collectively, these observations highlight the importance of the context (disease status)-specific transcriptional mechanisms during T cell differentiation.
Majority of studies demonstrate a dominant role of CD8
T cells in host defense. Features of CD8
T cell exhaustion and its effect on dysfunctional immune status have been extensively investigated [
]. Recent observations of CD4
T cell exhaustion in chronic viral infections suggest that CD4
T cells are also crucial for optimal infection control [
]. Most recently, Hwang et al. reported that Blimp-1 is upregulated and acts as a critical regulator for CD4
T cell exhaustion during chronic toxoplasmosis. Conditional deletion of Blimp-1 in CD4
T cells regained CD8
T cell function and improved infection control [
]. Contributions of CD4
T cell in leukemia are not well defined. Our findings demonstrate that, in addition to causing CD8
T cell dysfunction, Blimp-1 plays an equally important role in mediating CD4
T cell suppression in AML. Blimp-1 upregulates co-inhibitory receptors and associates with functional defect in both CD4
T cells. Interestingly, high Blimp-1 expression in CD4
, not CD8
T cells, correlates with high circulating leukemia blast (Table
), suggesting a potential unique contribution of CD4
T cell dysfunction in AML pathogenesis.
We thank all our patients for their trust, understanding, and willingness to provide their blood samples for our research.
This work was supported by the American Cancer Society Institutional Research Grant ACS IRG 124171-IRG-13-043-02 and the Kiesendahl Endowment funding (H Zheng). L.Z. is supported by the National Natural Science Foundation of China (No. 81671940).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.
LZ designed the experiments, performed the experiments, analyzed the results, made the figures, and wrote the manuscript. YK and BJ designed the experiments, performed the experiments, and analyzed the results. DFC, WCE, WBR, NDP, and MB acquired the samples and managed the patients. MW provided the biostatistics support. JZ helped with the sample collection. TDS and RJH reviewed the manuscript. HZ designed the experiments, analyzed the results, and wrote the manuscript. HZ conceived the concept, designed the experiments, oversaw the interpretation and presentation of the data, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was approved by the Institutional Review Board of Penn State University College of Medicine. Full informed consent was obtained from all patients.
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