Characterization of Immune Dysfunction and Identification of Prognostic 1
Immune-related Risk Factors in Acute Myeloid Leukemia 2
3
Authors: 4
Lu Tang1, 2, #
, Jianghua Wu1, 2, #
, Chenggong Li1, 2
, Huiwen Jiang1, 2
, Min Xu1, Mengyi Du
1, 2, 5
Zhinan Yin3, 4
, Heng Mei1, 2, *
, Yu Hu1, 2, *
6
Institutes: 7
1 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of 8
Science and Technology, Wuhan, 430022, Hubei, China 9
2 Hubei clinical medical center of cell therapy for neoplastic disease, Wuhan, 430022, Hubei, 10
China 11
3 Zhuhai Precision Medical Center, Zhuhai People's Hospital Affiliated with Jinan University, 12
Jinan University, Zhuhai, 519000, Guangdong, China 13
4 The Biomedical Translational Research Institute, Faculty of Medical Science, Jinan University, 14
Guangzhou, 510632, Guangdong, China 15
16
#First authors: Lu Tang and Jianghua Wu contributed equally to this study. 17
*Corresponding Authors: 18
Heng Mei*, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong 19
University of Science and Technology, No.1277 Jiefang Avenue, Wuhan 430022, Hubei, China; 20
Tel: +86-027-85726007; Fax: +86-027-85726387; E-mail: [email protected]; 21
Yu Hu*, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University 22
of Science and Technology, No.1277 Jiefang Avenue, Wuhan 430022, Hubei, China; Tel: 23
+86-027-85726007; Fax: +86-027-85726387; E-mail: [email protected]. 24
25
Running title 26
Immune profiling and its predictive utility in AML 27
Key words 28
Acute Myeloid Leukemia, Immune Dysfunction, Chemotherapy Response, Refractory/Relapsed, 29
Prognosis 30
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31
Conflicts of Interest Disclosure 32
The authors declare that they have no potential conflicts of interest. 33
34
Funding Support 35
This work was supported by grants from the National Natural Science Foundation of China (No. 36
81770132 for Yu Hu, and No. 81873434 for Heng Mei) and Major Technological Innovation 37
Special Project of Hubei Province of China (No. 2018ACA141 for Yu Hu). 38
39
Translational Relevance 40
Comprehensive immune profiling in newly-diagnosed AML patients suggests that T and NK cell 41
function defects are dominant aspects in immune dysfunction whereas B cell function remains 42
unaffected. T cell senescence and exhaustion, together with excessive NK maturation and 43
impaired γδ T cell function, are involved in immunosuppression that leads to evade anti-leukemia 44
immunity. Effective therapeutic response following chemotherapy correlates with T and NK 45
function restoration, and selective immune signatures significantly correlate with EFS and OS. 46
Although the cohort is small, it’s the first reported study that comprehensively and longitudinally 47
evaluates immune status in AML and facilitates our knowledge of predictive utility of 48
immunological biomarkers. Non-invasive immune testing of blood samples could be applied to 49
identify high risk for relapse, therapeutic reactivity and unfavorable prognosis, which greatly help 50
to guide clinical decisions in AML patients. 51
52
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Abstract 53
Purpose: This study aims to provide comprehensive insights into longitudinal immune landscape 54
in acute myeloid leukemia (AML) development and treatment, which may contribute to predict 55
prognosis and guide clinical decisions. 56
Experimental Design: Periphery blood samples from 79 AML patients (at diagnosis or/and after 57
chemotherapy or at relapse) and 24 healthy controls were prospectively collected. We performed 58
phenotypic and functional analysis of various lymphocytes through multiparametric flow 59
cytometry and investigated prognostic immune-related risk factors. 60
Results: Immune defects in AML were reflected in T and NK cells whereas B cell function 61
remained unaffected. Both CD8+ T and CD4
+ T cells exhibited features of senescence and 62
exhaustion at diagnosis. NK dysfunction was supported by excessive maturation and 63
downregulation of NKG2D and NKP30. Diseased γδ T cells demonstrated a highly-activated or 64
even exhausted state through PD-1 upregulation and NKG2D downregulation. Effective 65
therapeutic response following chemotherapy correlated with T and NK function restoration. 66
Refractory and relapsed patients demonstrated even worse immune impairments, and selective 67
immune signatures apparently correlated clinical outcomes and survival. PD-1 expression in 68
CD8+ T cells was independently predictive of poor overall survival (OS) and event-free survival 69
(EFS). 70
Conclusions: T cell senescence and exhaustion, together with impaired NK and γδ T cell function, 71
are dominant aspects involved in immune dysfunction in AML. Non-invasive immune testing of 72
blood samples could be applied to predict therapeutic reactivity, high risk for relapse and 73
unfavorable prognosis. 74
75
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Introduction 76
Acute myeloid leukemia (AML), a hematological malignancy with high heterogeneity, is the most 77
common leukemia among adults and usually associated with poor prognosis (1). Current risk 78
stratification is mainly based on conventional molecular and cytogenetic testing (2, 3). With the 79
advent of cancer immunotherapy, relevant exploration of risk stratification at the immune 80
level is crucial for personalized and precision therapy (4). To better predict prognosis and guide 81
clinical decisions, it is necessary to optimize the present risk stratification and management. Most 82
previous studies on improving prognosis in AML focused on the mechanisms of drug resistance, 83
with little attention given to the impacts of host immune status in disease development and 84
treatment. Chemotherapy, as a front-line treatment of AML, was reported to modulate T cell 85
function (5), and robust lymphocyte recovery after treatment predicted superior survival (6). How 86
immune microenvironment correlates with clinical response to chemotherapy and disease 87
progression remains great interests. 88
Successful anti-cancer immunity relies on the capacity of effector immune cells to recognize 89
and attack tumor cells and to alert other immune cells (7). Similar to solid tumors, AML is capable 90
of creating an immunosuppressive milieu, where both innate and adaptive immune responses are 91
profoundly deregulated (8). Much evidence suggests that AML blasts play role in the creation of 92
this dysfunction status through several unique immune evasion mechanisms (9, 10). Zhang’s 93
group have proved that elevated frequency of CD4+CD25
+CD127
low/- regulatory T (Treg) cells in 94
AML is associated to poor prognosis (11). Le Dieu et.al observed that circulating CD8+ T cells 95
showed abnormal phenotype and genotype at diagnosis, and formed defective immune synapses 96
with AML blasts (12). Moreover, AML blasts were reported to directly alter CD8+ T cells viability, 97
expansion, co-signaling and senescence marker expression in vitro and response to therapy 98
correlated with upregulation of costimulatory, downregulation of apoptotic and inhibitory 99
signaling pathways (13). Several studies have shown that CD8+ T cells expressing inhibitory 100
receptors are functionally impaired and predict AML relapse (14, 15). Natural killer (NK) and 101
natural killer-like T (NKT) cells demonstrated aberrant phenotype in AML and may impact 102
clinical outcome (7, 16). Immune interventions through facilitating early NK and gamma delta T 103
(γδ T) cell reconstitution may prevent relapse after HSCT (17). 104
However, comprehensive profiling of immunological signatures in AML development and 105
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treatment is still lacking, and little is known about how immune status correlates with 106
chemotherapy response and relapse. Here, we conducted a prospective study to perform 107
phenotypic and functional analysis of various lymphocytes (including CD4+ T, CD8
+ T, NK, NKT, 108
γδ T and B cells) to decipher the immune landscape in AML development and investigated 109
potential prognostic immune-related risk factors. 110
111
Materials and Methods 112
Study design and human specimens 113
Our study included 50 newly-diagnosed acute myeloid leukemia (ND-AML, 18-66 year) patients 114
and 24 healthy controls (HCs, 20-62 year). Blood samples from 24 patients achieved complete 115
remission (CR) after chemotherapy and 20 refractory and/or relapsed (RR) patients were also 116
collected for cross-sectional study. Paired pre- and post-chemotherapy peripheral blood (PB) 117
samples were collected from 15 patients. Basic characteristics of all AML patients included in our 118
study are summarized in Table 1 and Table S1. In accordance with 2016 World Health 119
Organization (WHO) classification, a diagnosis of AML is made based on the presence of ≥ 20% 120
blasts in bone marrow (BM) (1, 2). Treatment response after chemotherapy was assessed using 121
international standard criteria. CR is defined as < 5% blasts in BM with neutrophil counts ≥ 122
1000/μl and platelet counts ≥ 100000/μl (2). Refractory and relapsed is defined as patients who 123
fail to achieve CR after two courses of intensive chemotherapy or suffer relapse (2, 18). PB 124
samples were obtained from non-promyelocytic AML patients from the Department of 125
Hematology, Wuhan Union Hospital, China. This study was conducted in accordance with the 126
Declaration of Helsinki, and was approved by the Ethics Committee of Union Hospital, Tongji 127
Medical College, Huazhong University of Science, and Technology (# 2018/S475). Written 128
informed consents were provided to all participants prior to inclusion in this study. 129
130
Isolation of peripheral blood mononuclear cells 131
Fresh PB samples were collected in heparin-treated tubes from each subject and used for plasma 132
selection and peripheral blood mononuclear cells (PBMCs). After plasma selection, fresh PB 133
samples were diluted 1:1 with phosphate buffered saline (PBS) before separation of PBMCs by 134
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Ficoll-Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). Cells were washed 135
in RPMI 1640 supplemented with 10% fetal calf serum (FCS; PAA Laboratories), and then used 136
immediately for multiparametric flow cytometry. 137
138
Multiparametric flow cytometric analysis 139
For surface staining, PBMCs were washed twice in PBS containing 1% FBS (staining buffer), and 140
then were stained with fluorochrome-conjugated monoclonal antibodies (mAbs). Samples were 141
incubated with antibodies for 30 min at 4°C, then washed with staining buffer and kept at 4°C 142
until analysis. Intracellular staining for Foxp3, granzyme B (GZMB), perforin and CD107a was 143
performed after cell fixation and permeabilization (eBioscience), then intracellular proteins were 144
labeled with the corresponding mAbs conjugated with fluorescent molecules according to the 145
manufacturer instructions. List of all mAbs was shown in Supplementary Table S2. Flow 146
cytometry was performed on a BD LSRFortessa X-20 and data were analyzed with FlowJo V10 147
software (Tree Star). 148
149
Cytokines production assays 150
PBMCs were washed in RPMI1640 supplemented with 10% FCS (PAA Laboratories), and 151
cytokines production assays were performed after lymphocytes were stimulated with polymethyl 152
acrylate (PMA, 50ng/ml) and ionomycin (1µM) in the presence of Golgi-Stop. After 5 hours at 153
37°C, cells were first stained with fluorochrome-associated monoclonal antibodies specific for 154
surface molecules; next, cells underwent fixation and permeabilization for intracellular staining 155
with monoclonal antibodies specific for the following intracellular proteins: tumor necrosis 156
factor-α (TNF-α), interferon γ (IFN-γ), interleukin-2 (IL-2), interleukin-17A (IL-17A), and 157
interleukin-4 (IL-4). 158
159
Statistical analysis 160
Mann-Whitney U test was used to determine statistical difference between two groups and 161
Kruskal-Wallis test was used to determine statistical difference among three groups. Paired 162
samples were compared using Wilcoxon matched-pairs signed-rank test. Spearman’s rank 163
coefficient was used to determine correlations and non-linear regression (least squares ordinary fit) 164
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was also applied when plotted. Overall survival (OS) time and event-free survival (EFS) time 165
were calculated for survival analyses and median values were used for grouping the patients. 166
Kaplan-Meier log-rank test was used to compare between-group survival differences. Variables 167
with P
CD127 was downregulated in AML CD4+ T and CD8
+ T cells, which was an exhaustion feature in 195
many chronic viral infections as T cells might lose responsiveness to homeostatic cytokines (23). 196
Senescent T cells tend to lose co-stimulatory molecules such as CD27 and CD28 while expressing 197
CD57 progressively and irreversibly (24). Further characterization suggested that CD4+ T and 198
CD8+ T cells from AML patients showed downregulation in CD28, upregulation in CD57 but no 199
significant change in CD27. Generally, the higher expression of inhibitory receptors, the more 200
severe T cells are exhausted (25). Nevertheless, some researchers think that most PD-1high
CD8+ T 201
cells in healthy adult humans are effector memory cells rather than exhausted cells (26). Our data 202
revealed that diseased CD4+ T and CD8
+ T cells exhibited enhanced PD-1 expression, implying 203
highly activated or more exhausted state of T cells in patients. 204
Higher number of CD28-CD57
+PD-1
+ T subset reported in multiple myeloma (MM) was 205
associated with early relapse after HSCT, and this T cell clone negatively affect immunotherapy 206
(27, 28). Accumulated diseased CD4+ T and CD8
+ T cells co-expressed CD57 and PD-1, and 207
elevated percentages of the CD28-PD-1
+ phenotype were also found in AML patients 208
(Supplementary Fig. S2A-B). The total amounts of CD28-CD57
+ subset in CD8
+ T cells were 209
significantly increased when compared to HCs, implying the predominance of a senescent 210
phenotype (Supplementary Fig. S2C). Concomitantly, we observed the overall phenotype 211
CD28-CD57
+PD-1
+ accumulated in diseased CD8
+ T cells (P
immunoglobulin-like receptors (KIRs) and the heterodimeric C-type lectin receptor NKG2A (31), 225
which seemed unaffected in patients. Additionally, activating and inhibitory receptors expression 226
in NKT cells were similar for two groups (Fig. 1H). Lessened percentages of γδ T cells were 227
found in patients at diagnosis, especially Vδ2+ subsets. Furthermore, we observed increased PD-1 228
expression and decreased NKG2D expression in Vδ2+ cells, indicating highly-activated or even 229
exhausted states at diagnosis (Fig. 1I). 230
231
CD3+ T cells from AML patients show alteration of cytokines production 232
In next set of experiments, we assessed cytokines production of CD3+ T cells after stimulation 233
with PMA and ionomycin (Fig. 2A and Supplementary Fig. S3A). A further feature of T cell 234
exhaustion during chronic viral infections is the failure to produce effector cytokines in a 235
hierarchical manner, with the ability to produce IL-2 being lost at early stages of exhaustion, 236
followed by loss of TNF-α and finally IFN-γ (32). Unlike conventional T-cell exhaustion pattern 237
previously reported in chronic viral infections, CD4+ T, CD8
+ T and γδ T cells from AML patients 238
showed defective IFN-γ production but without significant reduction in the production of TNF-α 239
and IL-2. IL-4 production was also similar for patients and HCs. CD4+ T and γδ T cells from 240
patients demonstrated elevated expression of IL-17A, which was not seen in CD8+ T cells. The 241
differentiation of Th1 and Th17 cells were initially thought to be distinct and possibly antagonistic 242
(33). Compared with previous results using surface antigen markers, the IFN-γ/IL-17A ratio in 243
CD4+ T cells was consistent with Th1/Th17 ratio, indicating this antagonistic interaction in 244
patients. Unsupervised clustering analysis summarized in the heatmap of Fig. 2B also 245
demonstrated an alteration in cytokines production. The left part of the heatmap contains most of 246
the patient cohorts and T cells from patients shows obvious defective IFN-γ production but 247
enhanced IL-17A production. 248
Next, we investigated cytokines production in the overall CD28-CD57
+PD-1
+ T cells from 249
patients. The intensity of IFN-γ and TNF-α expression (P
expression correlated with cytokine production in terminally senescent CD28-CD57
+ 255
subpopulation (Fig. 2D) and found it negatively correlated with TNF-α and IFN-γ expression. 256
Although AML CD8+ T cells show highly senescent state, elevated PD-1 expression may explain 257
the finding of overall lower production of IFN-γ. Further functional characterization demonstrated 258
no obvious alteration of degranulation capacity and cytotoxic molecules expression (CD107a, 259
GZMB and perforin) in total T and NK cells between patients and HCs (Supplementary Fig. 260
S3B). 261
262
Immune signatures diverged among patients with different therapeutic response and relapse 263
Given the importance of AML blasts in influencing immune signatures, we hypothesized that a 264
change in the leukemia burden and hematopoietic milieu could affect this dysfunction state. Paired 265
comparisons of immune features were conducted in 15 patients (10 achieved CR, 5 failed to 266
achieve CR) before and after induction chemotherapy (Supplementary Fig. S4). To avoid 267
potential bias caused by chemotherapy regimens, only patients who received standard induction 268
regimens (anthracycline and cytarabine “3+7”) were included in this pairwise comparison. When 269
analyzed by therapeutic response, several immune features changed only in CR patients compared 270
with pre-treatment levels (Fig. 3A). Overall, the frequency of CD3+ T lymphocytes was increased 271
after achieving CR, which may be owing to the elimination of blasts. The percentages of CD8+ 272
TNaïve (P=0.0273) and CD8+ TCM (P=0.0116) subsets were significantly higher in responders 273
versus non-responders. CD28 was restored in CD8+ T cells while CD127 was restored in CD4
+ T 274
cells. Excessive NK cell maturation and NKG2D expression in Vδ2+ T cells (P=0.0059) were also 275
improved at the time of achieving CR. Moreover, we confirmed PD-1 downregulation in CD8+ T 276
(P=0.0254) and Vδ2+ T cells (P=0.0059) following effective treatment. 277
Furtherly, we extended studies to analyze the difference in immune signatures between CR 278
group (achieved stable CR after chemotherapy, n = 24) and RR group (failed to achieved CR after 279
chemotherapy or suffered a relapse, n = 20) (Fig. 3B). CD8+ T cells in CR group demonstrated 280
higher percentages of TNaïve subsets and relatively lower percentages of terminally differentiated 281
effector T subset (TEMRA). CD4+ and CD8
+ T cells from RR group showed higher PD-1 expression 282
(P=0.0201 and P=0.0006, respectively). In-depth analysis revealed decreased CD8+CD28
+ T cells, 283
increased CD8+CD57
+ T cells and concomitantly increased CD8
+CD28
-CD57
+ T cells (P=0.0261) 284
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in RR group, implying CD8+ T cells of higher senescence states in refractory patients or patients at 285
relapse. Additionally, patients in RR group demonstrated excessive NK maturation and defective 286
γδ T immunity. 287
288
289
Correlations between immunological signatures and clinical characteristics 290
Due to observed heterogeneity with the analyzed markers, we investigated whether immune 291
signatures could be associated with clinical characteristics. Overall results of correlation analyses 292
based on Spearman’s rank coefficient test were presented in Supplementary Table S3, and we 293
next focused on three meaningful clinical parameters that proved to be predictive of prognosis in 294
following regression analysis: age, white blood cell count and cytogenetic risk group (Table 2). 295
Interestingly, the age was positively related to CD28-CD57
+, PD-1
+ and CCR7
-CD8
+ T subset 296
frequency (Fig. 4A), implying CD8+ T cell terminal differentiation and exhaustion in elderly 297
patients. Hyperleukocytosis usually indicate higher leukemia burden in AML patients, which may 298
account for the negative correlation with CD3+ T frequency and Th1/Th17 ratio (Fig. 4B). 299
Additionally, patients with elevated white blood cell counts demonstrated NKG2D and NKP46 300
downregulation in NK cells (Table S3). More importantly, we observed significant difference of 301
terminal senescent CD8+ T cells among patients with different cytogenetic prognostic risk based 302
on ELN guideline; patients with adverse prognostic factors demonstrated higher percentages of 303
CD28-CD57
+CD8
+ T cells (Fig. 4C). 304
305
Predictive utility in chemotherapy response, relapse and survival 306
Patients demonstrated survival divergence among conventional cytogenetic low-risk, 307
intermediate-risk and high-risk groups (Fig. 4D). Further survival analysis suggested that selective 308
immune signatures directly correlated with OS and EFS when assessed as categorical variables 309
(Supplementary Table S4). Pre-treatment PD-1+CD8
+ T and CD28
-CD57
+CD8
+ T subsets were 310
related to poor OS (HR = 4.60, P=0.0087 and HR = 4.28, P=0.0125, respectively) and EFS (HR = 311
4.10, P=0.0094 and HR = 4.96, P=0.0030, respectively) from diagnosis (Fig. 4E). Elevated day 312
+30 post induction chemotherapy PD-1+CD8
+ T frequency correlated with unfavorable OS (HR = 313
6.84, P=0.0437) and EFS (HR = 6.39, P=0.0190) after induction chemotherapy (Fig. 4F). 314
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Moreover, increased proportions of day +30 post induction chemotherapy immature NK cells were 315
associated with improved EFS (HR = 0.13, P = 0.0086), but not with OS (Table S4). In univariate 316
Cox regression analysis of OS and EFS, we screened out five significative prognostic risk factors: 317
age, pre-treatment PD-1+CD8
+ T and CD28
-CD57
+CD8
+ T frequency as continuous variables; 318
ELN risk group (adverse, intermediate and favorable) and WBC count (45G/L) 319
as categorical variables (Table 2). From multivariate Cox regression analyses results, conventional 320
cytogenetical risk and hyperleukocytosis (WBC count >45G/L) were independent risk factors of 321
OS and EFS. Age and pre-treatment PD-1 expression in CD8+ T cells were only independent risk 322
factors of OS but not EFS. Survival difference in OS and EFS was not independent of 323
pre-treatment CD28-CD57
+CD8
+ T frequency, which may be on account of its correlation with age 324
and cytogenetic risk group. 325
326
Discussion 327
Certain chemotherapies and targeted agents for cancer can exert their anti-tumor effects at least in 328
part through immune activation (35). Immune microenvironment plays a key role in anti-leukemia 329
effect, and long-term survival of AML patients may be improved through modulating immune 330
impairments. Our findings suggest that immune defects are operative in T and NK cells that are 331
important components of anti-tumor immunity whereas B cell function remains unaffected in 332
AML, and immunological signatures may be novel prognostic biomarkers in leukemia. 333
T cell dysfunction in cancer displays functional unresponsiveness, including senescence, 334
exhaustion, anergy, and self-tolerance (36-38). To date, much emphasis has been placed on CD8+ 335
T cell dysfunction (13, 20). Although CD4+ T cells show less senescent level than CD8
+ T cells, 336
our study reveals that both CD8+ T and CD4
+ T cells from AML patients exhibit features of 337
senescence and exhaustion at diagnosis. Replicative senescence is the natural age-related process 338
that occurs with a shortening of telomeric ends, but premature senescence is a 339
telomere-independent senescence induced by outside factors such as cellular stress (24, 39). T cell 340
exhaustion is a poor responsive status with upregulated expression of inhibitory receptors, 341
decreased production of effective cytokines, and reduced cytotoxic activity (40). Circulating T 342
cells from AML patients demonstrate signatures of terminal senescence 343
(CD28low
CD57high
IFN-γhigh
TNF-αhigh
) and exhaustion (PD-1high
IFN-γlow
TNF-αlow
) simultaneously. 344
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Senescent CD28-CD57
+ T cells show higher capacities for IL-2, IFN-γ and TNF-α production, but 345
exhausted T cells have diminished cytokine production and effector function (37). AML patients 346
show poor IFN-γ production in overall T cells despite of obvious presence of senescence, which 347
could be explained by elevated PD-1 expression (37). More importantly, these terminally 348
senescent and exhausted T cells could persist with recruitment in RR patients, indicating even 349
worse T cell dysfunction at poor therapeutic response or relapse. 350
In context of the existence of AML blasts in PB, T cells may encounter a complicated network 351
that suppresses their immune effectiveness. As Treg cells control peripheral immune tolerance, 352
CD4+ helper and CD8
+ cytotoxic T cells could be forced by Treg cells into T cell senescence by 353
inducing DNA damage using metabolic competition during cross-talk (41, 42). Consistent with 354
previous studies, we detected similarly accumulating Treg cells in AML (11), which may 355
accelerate T senescence and exhaustion. IL-17A exhibits pro-tumor effects through promoting the 356
proliferation of IL-17 receptor-positive AML cells and inhibit the generation of Th1 cells (43). 357
Therefore, another important link in this suppressive network may be the unbalance between Th1 358
and Th17, especially patients with high leukemia burden. 359
NK and γδ T cells provide first-line defense against virus-infected cells and tumors, and their 360
function is governed by a balance between inhibitory and activating receptors (44, 45). Lessened 361
frequency of NK, NKT and γδ T cells indicates slack innate immunity in AML. One important 362
parameter involved in NK cell dysfunction is the excessive maturation observed in our study as 363
well as the previous report (46). NK cell defects were further supported by downregulation of 364
activating receptor NKG2D and NKP30, which may weaken the recognition and interaction 365
between NK cells and AML blasts. Meanwhile, diseased γδ T cells demonstrate a highly-activated 366
or even exhaustion state through a profound PD-1 upregulation and NKG2D downregulation. T 367
and NK cells from AML patients retain normal degranulation and GZMB and perforin production, 368
implying this was not a predominant factor in T and NK defects. Conclusively, these results 369
suggest that AML blasts may induce long-lasting dysfunction in T and NK cells and favor 370
leukemia survival. 371
New findings have emerged from our data that immune reconstitution occurs following 372
chemotherapy with therapeutic response. T and NK cell function restoration is principally 373
reflected in recovered proportions, decreased extent of terminal differentiation and partly 374
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improved T cell senescence and exhaustion. Patients in RR group exhibit selectively higher 375
senescent and exhausted CD8+ T (CD28
low/CD57
high/PD-1
high) and CD4
+ T (PD-1
high) cells, less γδ 376
T cells and excessive NK cell maturation. Moreover, our study reveals that these specific immune 377
signatures apparently correlate with OS and EFS in AML patients. Terminally senescent and 378
exhausted CD8+ T (CD28
low/CD57
high/PD-1
high) cells observed at diagnosis prove to be associated 379
with poor prognosis, and this dysfunction status persisting even after induction chemotherapy also 380
indicates worse long-term survival. More importantly, PD-1+CD8
+ T is confirmed to be 381
independent risk factor of poor prognosis in further multivariate Cox regression analysis, which 382
makes us more alert to CD8+ T exhaustion in AML development and treatment. These interesting 383
findings imply that non-invasively immune-based biomarkers may be novel prognostic risk 384
factors. 385
Immunotherapy remains a highly promising approach for the treatment of AML patients, 386
particularly those otherwise ineligible for HSCT or at relapse. Although conventional cytogenetic 387
risk stratification could identify patient subgroups with different survival possibilities, it’s 388
guidance role in treatment decisions is far from being desired, especially in immunotherapies. 389
Patients with favorable risk may also demonstrate immune impairments to some extent. 390
Therefore, dynamic monitoring of immune status is crucial for personalized 391
immune-modulating therapies to enable better clinical outcomes (4, 10). Potential approach to 392
enhance anti-leukemia is to improve T and NK dysfunction (22, 24, 47-50), such as PD-1 inhibitor, 393
chimeric antigen receptor T cell therapy and NK or γδ T-based adoptive immunotherapies. 394
Purposeful therapeutic strategies during treatment decisions could be made according to the 395
immune impairments in each patient. Nevertheless, determining the optimal immunotherapy and 396
best timing in relation to chemotherapy and HSCT remains to be solved. Future efforts are needed 397
to delineate how best to integrate personalized immunotherapies into curative treatment regimens 398
for AML. 399
Our studies still have several limitations. First, phenotypic sculpting of AML blasts through 400
immunoediting needs further investigation because immune suppression refers to the interaction 401
between cancer and immune system. Second, the mechanism how AML blasts induce immune 402
dysfunction is insufficient due to extensive alteration across T and NK cells and the complexity in 403
identifying antigen-specific cytotoxic immune cells. In addition, we need to further validate their 404
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predictive value of response to tailored immunotherapies rather than overall prognostic value to 405
better guide immunotherapy decisions. Finally, heterogeneity is moderately obvious in 406
immunological markers due to limited sample size, and clinical consequences of such 407
observations should be checked up with larger cohorts of patients. 408
In conclusion, this is the first study longitudinally deciphering comprehensive immune 409
landscape in AML patients during the course of chemotherapy. T cell senescence and exhaustion, 410
together with impaired NK and γδ T cell function, are involved in immune dysfunction in AML 411
development, which are improved to some extent following effective therapeutic response. 412
Remarkably, we firstly demonstrate that non-invasive immune testing of blood samples could be 413
applied to identify high risk for relapse, therapeutic reactivity and unfavorable prognosis in AML. 414
415
Acknowledgements: 416
This work was supported by grants from the National Natural Science Foundation of China (No. 417
81770132 for Yu Hu, and No. 81873434 for Heng Mei) and Major Technological Innovation 418
Special Project of Hubei Province of China (No. 2018ACA141 for Yu Hu). We thank all 419
researchers’ contributions, as well as the leukemia patients and healthy participants. 420
421
Authors' contributions 422
Conception and design: Y. Hu, H. Mei, Z.N. Yin 423
Development of methodology: Y. Hu, H. Mei, Z.N. Ying, L. Tang 424
Acquisition of data (provided animals, acquired and managed patients, provided facilities, 425
etc.): H. Mei, L. Tang, J.H. Wu, C.G. Li, W.H. Jiang, M. Xin, M.Y. Du 426
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational 427
analysis): H. Mei, L. Tang, J.H. Wu 428
Writing, review, and/or revision of the manuscript: H. Mei, L. Tang, J.H. Wu 429
Administrative, technical, or material support (i.e., reporting or organizing data, 430
constructing databases): Y. Hu, H. Mei, Z.N. Yin 431
Study supervision: Y. Hu, H. Mei 432
433
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Tables and Figures
Table 1 Basic characteristics of AML participants.
Table 2 Univariate and multivariate Cox regression analysis of OS and EFS.
Fig. 1 Lymphocyte composition and immunophenotypic characterization of T and NK cells.
Fig. 2 Functional characterization of cytokine production in CD4+ T, CD8
+ T and γδ T cells.
Fig. 3 Immune signatures diverged among patients with different therapeutic response and relapse.
Fig. 4 Correlation and survival analysis of clinical parameters and immune signatures.
Table 1 Basic characteristics of AML participants
Patients ND-AML CR RR
Included 50 24 20
Sex (females/males) 23/27 12/12 11/9
Age (median and range) 40(18-66) 40(19-62) 44(21-57)
FAB type
M0 0 (0.0%) 0 (0.0%) 0 (0.0%)
M1 14 (28.0%) 6 (25.0%) 7 (35.0%)
M2 20 (40.0%) 9 (37.5%) 9 (45.0%)
M4 11 (22.0%) 7 (29.2%) 3 (15.0%)
M5 3 (6.0%) 2 (8.3%) 0 (0.0%)
M6 2 (4.0%) 0 (0.0%) 0 (0.0%)
M7 0 (0.0%) 0 (0.0%) 1 (5.0%)
Cytogenetic risk
Favorable 19 (38.0%) 12 (50.0%) 4 (20.0%)
Intermediate 23 (46.0%) 11 (45.8%) 9 (45.0%)
Adverse 8 (16.0%) 1 (4.2%) 7 (35.0%)
WBC count (G/L)
Median and range 9.25 (1.10-85.44) 4.90 (0.92-18.88) 5.74 (0.80-79.33)
< 10 26 (52.0%) 16 (66.7%) 9 (45.0%)
10-45 18 (36.0%) 8 (33.3%) 6 (30.0%)
> 45 6 (12.0%) 0 (0.0%) 5 (25.0%)
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Table 2 Univariate and multivariate Cox regression analyses of OS and EFS
Univariate cox regression Multivariate cox regression
HR 95% CI p Value HR 95% CI p Value
low up low up
0S Cytogenetic risk 0.004 0.009
Favorable 1.000 1.000
Intermediate 5.586 0.650 47.999 0.117 0.295 0.011 7.761 0.465
Adverse 23.017 2.735 193.737 0.004 6.460 0.568 73.517 0.133
WBC count (G/L) 0.006 0.002
45 7.011 1.933 25.427 0.003 69.573 6.355 761.714 0.001
Age (years) 1.087 1.034 1.143 0.001 1.121 1.009 1.245 0.033
CD8+PD-1
+ (%) 1.064 1.025 1.106 0.001 1.092 1.011 1.180 0.025
CD8+CD28
-CD57
+ (%) 1.040 1.012 1.068 0.005 1.004 0.950 1.061 0.887
EFS Cytogenetic risk 0.007 0.023
Favorable 1.000 1.000
Intermediate 9.807 1.223 78.642 0.032 2.037 0.172 24.127 0.573
Adverse 26.784 3.129 229.289 0.003 10.315 0.967 109.998 0.053
WBC count (G/L) 0.021 0.008
50 4.397 1.309 14.763 0.017 9.934 2.171 45.455 0.003
Age (years) 1.082 1.033 1.134 0.001 1.072 0.998 1.151 0.056
CD8+PD-1
+ (%) 1.067 1.028 1.107 0.001 1.029 0.976 1.084 0.286
CD8+CD28
-CD57
+ (%) 1.041 1.015 1.067 0.002 1.015 0.972 1.059 0.509
Main Figure legends:
Fig. 1 Lymphocyte composition and immunophenotypic characterization of T and NK cells.
T and NK function defects are dominant components involved in immune dysfunction in AML.
Circulating lymphocytes from ND-AML patients (n = 50) and HCs (HCs) (n = 24) were analyzed by
multiparameter flow cytometry. (A) Scatter plots of T, NK and B cell frequency; (B) scatter plots of
regulatory T cells frequency; (C) relative proportion of Th, Tfh and Tc subsets (histograms); (D) flow
cytometry gating by CD45RA and CCR7, and histograms of proportions of TNaïve, TCM, TEM, TEMRA
subsets in CD4+ T and CD8+ T cells (mean±SEM); (E)-(F) histograms of CD127, CD27 CD28, CD57
and PD-1 expression in CD4+ T and CD8+ T cells (median, min to max); Pretreatment circulating NK,
NKT and γδ T cells from ND-AML patients (n = 29) and HCs (n = 24) were analyzed by
multiparameter flow cytometry: (G)-(H) histograms of NKG2D, NKP30, NKP46, NKG2A and KIR
expression in NK and NKT cells (median, min to max); (I) histograms of NKG2D and PD-1 expression
in Vδ2+ T cells (median, min to max). Mann-Whitney U test was used to determine statistical
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difference between two groups.
Fig. 2 Functional characterization of cytokine production in CD4+ T, CD8+ T and γδ T cells.
Circulating CD4+ T, CD8+ T and γδ T cells from ND-AML patients (n = 20) and HCs (n = 9) were
analyzed by multiparameter flow cytometry. (A) Histograms of IFN-γ and IL-17A production in CD4+
T, CD8+ T and γδ T cells (median, min to max; Mann-Whitney U test ); (B) heatmap with unsupervised
clustering analyses (Morpheus software); (C) comparison of cytokines production between
CD28-CD57+ T and non CD28-CD57+ T cells (gated in CD3+ T cells from patients) and corresponding
histograms (mean±SEM, Wilcoxon matched-pairs signed-rank test); (E) comparison of cytokines
production between PD-1- and PD-1+ subsets (gated in CD3+CD28-CD57+ T cells from patients) and
corresponding histograms (mean±SEM, Wilcoxon matched-pairs signed-rank test).
Fig. 3 Immune signatures diverged among patients with different therapeutic response and relapse.
(A) Pairwise comparisons between pre- and post-chemotherapy immune signatures from 15 AML
patients (10 patients achieved CR, 5 patients failed to achieve CR) using Wilcoxon matched-pairs
signed-rank test; (B) scatter plots (mean±SEM) of post-treatment immune signatures between CR
group (n =24) and RR group (n = 20) using Mann-Whitney U test.
Fig. 4 Correlation and survival analysis of clinical parameters and immune signatures.
(A)-(B) Correlation analysis between several immune signatures with age and WBC count using
Spearman’s rank coefficient and corresponding non-linear regression plots (least squares ordinary fit);
(C) comparisons of immune signatures among patients with different cytogenetic risk using
Kruskal-Wallis test and corresponding scatter plots (mean±SEM); Survival analyses (OS and EFS)
were performed using log-rank (Mantel–Cox) test and curves were plotted using Kaplan-Meier method,
and corresponding error bars show 95% CI: (D) patients demonstrated survival divergence among
cytogenetic low-risk, intermediate-risk and high-risk groups (n = 39); (E) increased pre-treatment
PD-1+CD8+ T and CD28-CD57+CD8+ T subtypes were related to poor OS and EFS from diagnosis (n =
39); (F) increased day+30 post-induction chemotherapy PD-1+CD8+ T subtypes were related to poor
OS and EFS after induction chemotherapy (n = 15). Low and high indicate below or above median
values.
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Published OnlineFirst January 7, 2020.Clin Cancer Res Lu Tang, Jianghua Wu, Cheng-Gong Li, et al. LeukemiaPrognostic Immune-related Risk Factors in Acute Myeloid Characterization of Immune Dysfunction and Identification of
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