Expression of immune inhibitory receptor ILT3 in acute myeloid leukemia with monocytic differentiation


  • How to cite this article: Dobrowolska H, Gill KZ, Serban G, Ivan E, Li Q, Qiao P, Suciu-Foca N, Savage D, Alobeid B, Bhagat G, Colovai AI. Expression of immune inhibitory receptor ILT3 in acute myeloid leukemia with monocytic differentiation. Cytometry Part B 2013; 84B: 21–29.



The diagnosis of AML with monocytic differentiation is limited by the lack of highly sensitive and specific monocytic markers. Immunoglobulin-like transcript 3 (ILT3) is an immune inhibitory receptor expressed by myelomonocytic cells and at high levels by tolerogenic dendritic cells.


Using flow cytometry, we analyzed the expression of ILT3 in 37 patients with AML and 20 patients with no detectable disease.


We showed that ILT3 was expressed in all cases of AML displaying monocytic differentiation (FAB M4/M5; N = 18), but not in AML M1/M2 and M3 (N = 19; P < 0.0001). Co-expression of ILT3 and immature cell markers, such as CD34 and CD117, was observed in monoblastic leukemia. ILT3 expression was preserved after treatment in M4/M5 patients with refractory or relapsed disease. ILT3 expression was associated with the presence of cytogenetic abnormalities linked to an intermediate prognosis (P = 0.001). Rare CD45dimCD34+CD117+ILT3+ cells were identified in noninvolved bone marrow, suggesting that ILT3 expression is acquired at an early stage by normal myelomonocytic precursors.


ILT3 is a highly sensitive and specific marker which distinguishes AML with monocytic differentiation from other types of AML. Testing of ILT3 expression should be incorporated into the initial diagnostic work-up and monitoring of patients with AML. © 2012 International Clinical Cytometry Society


Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults (1,2). Despite major advances in our understanding of the biology of AML, the five-year survival of AML patients is only 20–40%. It has been proposed that AML originates from self-renewing hematopoietic stem cells (HSC)/progenitors that have acquired multiple genetic and/or epigenetic changes (3). These cells initiate a developmental hierarchy of single- or multiple lineage precursors exhibiting various degrees of maturation arrest. The heterogeneity of AML is evident from the wide variety of clinical manifestations, phenotypic features, molecular and cytogenetic alterations, and response to therapy (4–6).

In clinical practice, the accurate diagnosis of AML subtypes is essential for risk stratification and treatment planning. The World Health Organization (WHO) and the French-American-British (FAB) classification systems are most commonly used to subtype AML. Although the WHO classification system relies heavily on cytogenetics and molecular data, these data are often not available at the time of diagnosis (1,7). Furthermore, cytogenetic abnormalities are absent in over 40% of the patients with AML (5, 6, 8) The absence of any chromosomal alterations is, in fact, the most frequent cytogenetic feature seen in patients with AML. Therefore, characterization of cellular markers using expeditious tools, such as flow cytometry and immunohistochemistry, has great practical use for subtyping of AML.

AML with monocytic differentiation includes FAB M4, M5a, and M5b subtypes and shows distinct clinical features, such as high risk of extramedullary disease, high leukocyte count, and coagulation abnormalities (9,10). Certain translocations, such as t(9;11) and 16q22, involving the mixed lineage leukemia (MLL) and the core-binding factor beta genes, respectively, are commonly seen in monocytic leukemias (2,11). Mutations of the nucleophosmin (NPM1) gene have been also associated with myelomonocytic or monocytic morphology and are predictive of favorable outcome (6). However, these abnormalities are not absolutely specific for the monocytic lineage and identify only a fraction of patients with AML displaying monocytic differentiation.

Immunohistochemical analysis is often used to subtype AML yet its value in the diagnosis of AML with monocytic components is limited by the lack of highly sensitive and specific monocytic markers (12). For example, CD68, a marker used for identification of monocytes or macrophages, is also expressed by normal neutrophils and a number of nonmonocytic AMLs (13). The hemoglobin scavenger receptor CD163 has increased specificity for the monocytic lineage compared with CD68 yet it has low sensitivity (14,15). Several other cell markers are being used for ascertaining monocytic differentiation by flow cytometry, i.e., CD4, CD11c, CD14, CD36, and CD64 (16–18). Although the expression of these markers by the leukemic cells is helpful for lineage assignment, the diagnosis of AML with monocytic differentiation remains challenging.

The inhibitory receptor ILT3 is a member of the immunoglobulin-like transcript (ILT, LIR or LILR) family and is expressed by dendritic cells, monocytes, endothelial cells and osteoclasts, but not by lymphocytes (19–21). Encoded in the leukocyte receptor cluster on human chromosome 19, the ILT proteins are structurally and functionally related to the killer cell immunoglobulin-like receptors (KIR) and deliver either activating or inhibitory signals (22–24). Inhibitory ILT proteins display intracytoplasmic ITIM motifs that recruit SHP-1 and SHIP-1 phosphatases, which transmit downstream inhibitory signals. In contrast, activating ILTs have short cytoplasmic tails and associate with adaptor proteins, such as FceRIg, to activate cell signaling (25).

The function of ILT proteins expressed by antigen-presenting cells (APC), such as monocytes and dendritic cells, has been extensively documented (26–29). Dendritic cells expressing high levels of inhibitory receptors ILT3 and ILT4 were shown to induce energy of CD4+ T helper cells and differentiation of CD8+ T suppressor cells (23, 26, 27). In contrast, knockdown of ILT3 renders dendritic cells more sensitive to TLR signals (27). We have previously shown that ILT3 is a useful marker for the identification of aggressive chronic lymphocytic leukemia (CLL) (30). Although absent on normal B lymphocytes, ILT3 is expressed by leukemic B cells from a subset of CLL patients displaying extensive lymph node involvement.

In this study, we investigated ILT3 expression by normal and leukemic myeloid precursors. We report that ILT3 expression identifies normal hematopoietic precursors committed to the monocytic lineage and that ILT3 is a reliable marker that distinguishes AML with monocytic differentiation from other types of AML.


Case Selection

A total of 61 specimens, which included 52 bone marrow (BM) and nine peripheral blood (PB) samples, were analyzed between January 1, 2003, and December 31, 2006, according to a protocol approved by the IRB of Columbia University (Fig. 1). Samples were obtained for diagnostic purposes from a total of 57 patients followed at our institution. Diagnosis was established based on morphology, immunohistochemistry, flow cytometry, and cytogenetics analysis (1,7). After the diagnostic work-up had been completed, ILT3 expression was evaluated in left-over samples using flow cytometry. Since the inclusion of study cases was limited by the availability of leftover cells, the enrollment of all cases referred for diagnostic work-up was not possible. Thus, the frequency of certain AML subtypes within the studied cases may not reflect the incidence of those subtypes within the general patient population.

Figure 1.

Chart of patient specimens. BM: bone marrow; PB: peripheral blood.

Out of 61 specimens, 41 were obtained from patients with AML (Fig. 1). Paired PB and BM samples were available in two patients. The majority of the samples were obtained from patients newly diagnosed with AML (30 out of 41), while 11 out of 41 samples were from patients with relapsed or refractory disease. For the purpose of this study, leukemia cases were categorized according to the FAB criteria (1, 7, 31). Cases of FAB M4, M4Eo, M5a, and M5b were considered as displaying monocytic differentiation, while FAB M0, M1, M2, M3 (acute promyelocytic leukemia, APL), M6, and M7 were not. Three out of nine APL cases were classified as microgranular variants based on the absence or paucity of granules and predominantly bi-lobed nuclear shape (1). The assessment of monocytic differentiation and designation of M4/M5 subtypes were based on cytomorphology (1,7). The differential count of mature monocytes, promonocytes, and monoblasts was performed on air-dried, Giesma-stained, aspirate smear slides of optimal quality. The following features identified by flow cytometry were considered to support monocytic differentiation: expression of markers associated to the monocytic lineage, e.g., CD4, CD11c, CD14, or CD64, low or absent myeloperoxidase (MPO), and intermediate side scatter (SSC). Cytogenetics data were available in 32 out of 37 patients with AML.

The remaining 20 samples were obtained from patients with no evidence of bone marrow involvement, as established by clinical evaluation and morphology, flow cytometry and cytogenetics analysis. Thirteen of the noninvolved samples were obtained from patients previously treated for AML (three AML without differentiation, five APL, three AML with monocytic differentiation, two undefined AML types), while the remaining seven samples were obtained from patients with other diseases (one aplastic anemia, two gastric B-cell lymphoma, two acute lymphocytic leukemia in remission, and two viral infections).

Clinical Parameters

The following clinical parameters were analyzed in relation to ILT3 expression by leukemic cells in patients with AML: age, gender, AML type, WBC, PB monocyte count, hemoglobin level, platelet count, frequency of blasts in PB and BM, chromosomal abnormalities, and overall survival.

Flow Cytometry

Immunophenotypic characterization of BM and PB samples was performed using three-color flow cytometry, as previously described (30). The antibody combinations were selected to permit identification of the lineage and maturation state of the ILT3-expressing cells (Table 1). ILT3-PC5 and IgG PC5 antibodies were obtained from Beckman Coulter (Miami, FL). All the other antibodies were obtained from BD BioScience (San Jose, CA). The cut-off between ILT3 positive and negative cells was set using an isotype control antibody (IgG PC5).

Table 1. Antibody Combinations and Corresponding Fluorochromes Used for Immunophenotypic Analysis
12CD2CD7 CD45
13CD3CD16+56 CD45
15CD34CD117 CD45
16CD64MPO CD45

All samples were tested within 48 h of collection. Cells were run and analyzed on an FACSCalibur (BD Biosciences) using CellQuestPro software. Leukemic cells were considered positive for any given marker if >10% of the cells expressed that marker.

Statistical Analysis

The relationship between ILT3 expression and clinical parameters was studied using the Chi-square test, Student's t test of significance, and multiple regression analysis (IBM SPSS Statistics 17.0).


To characterize the expression of inhibitory receptor ILT3 on normal and neoplastic hematopoietic precursors, we analyzed 20 BM samples obtained from patients with no evidence of neoplastic disease and 41 specimens obtained from patients with AML. Flow cytometric analysis of noninvolved BM indicated that a high proportion of the CD14+ monocytes, specifically 80 ± 9%, expressed ILT3 (Fig. 2), while granulocytes were essentially negative. This profile is similar to that previously reported for circulating myelomonocytic cells (19,30). Within the CD45dim/low side scatter (SSC) gate, which includes hematopoietic precursors, the frequency of ILT3+ cells was 10 ± 7% of the gated cells (0.4 ± 0.3% of all nucleated cells; Fig. 2). There was no significant difference between the frequency of CD45dim/ILT3+/low SSC cells identified in the BM samples obtained from AML patients in remission at the time of testing (10 ± 9%; N = 13) and the frequency of CD45dim/ILT3+/low SSC cells from non-AML patients (8 ± 3%; N = 7). CD45dim/ILT3+/low SSC cells co-expressed CD33 and included subpopulations expressing CD34, CD117 (c-kit), and CD14 (Fig. 2). Co-expression of ILT3 and CD19 was not observed, indicating that B cell precursors do not express ILT3 (data not shown). As illustrated in Figure 2, ILT3 positive cells included CD14+ and CD14- cells, consistent with a maturation pattern in which ILT3 expression by BM monocytic cells is acquired prior to CD14 expression. Co-expression of ILT3 and the early markers CD34 and CD117 suggests that ILT3 is acquired at an early step of hematopoietic differentiation.

Figure 2.

Flow cytometric analysis of a whole bone marrow aspirate obtained from a patient with no evidence of hematological disease. Cells were gated as follows: R1 (CD45dim/low to intermediate SSC): includes precursor cells; R2 (CD45bright/intermediate SSC): monocytes; R3 (CD45bright/low SSC): lymphocytes; R4 (CD45dim/high SSC): granulocytes; Cell surface expression of ILT3 and CD14, CD33, CD34 or CD117 is depicted. The results are representative for the immunophenotypic profile observed in 20 noninvolved bone marrow samples. [Color figure can be viewed in the online issue which is available at]

In patients with AML, leukemic blasts accounted for 22–93% of the nucleated cells, with a mean of 52 ± 34% and 25 ± 32% in BM and PB samples, respectively (Table 2). Flow cytometric analysis indicated that ILT3 was variably expressed by the leukemic cells (range 1–99%; mean ± SD, 44 ± 41%). However, the frequency of ILT3 positive cells in patients with AML displaying monocytic differentiation (M4/M5) was significantly higher than that observed in patients with other forms of AML, namely 65 ± 33% versus 1 ± 1% of the leukemic cells (P < 0.0001) (Table 2). The best correlation between ILT3 expression and the AML type (P < 0.0001) was obtained using a cut-off of 10% of cells expressing ILT3. Using this cut-off, all of the 18 cases of AML with monocytic differentiation were ILT3 positive (ILT3+ cells >10%), and all non-monocytic AML cases were ILT3 negative (ILT3+ cells ≤10%; P < 0.0001; Table 2). Small populations of ILT3+ cells, accounting for less than 10% of the CD45dim cells, were detected in some non-monocytic AML cases. Although these cells may represent a minor monocytic component of the leukemia, none of these cases relapsed as monocytic forms. The relatively limited number of cases enrolled in this study did not allow us to test the threshold of 10% ILT3+ cells in a separate validation group. However, due to the highly significant correlation between AML type and ILT3 expression using a cut-off of 10% ILT3 expressing cells, we have applied this cut-off throughout the study. Paired BM and PB samples had similar frequencies of ILT3+ cells among the leukemic cells (data not shown).

Table 2. Clinical Parameters and ILT3 Expression by Leukemic Cells from Patients with AML
Group variableAll patientsILT3 positive (>10% of leukemic cells)ILT3 negative (≤10% of leukemic cells)p valuea
  • a

    Chi-square analysis was applied to study the relationship between ILT3 expression and gender, AML type, or cytogenetic abnormalities. To study the relationship between ILT3 and continuous variables, such as age, WBC, frequency of various cell subsets or hemoglobin, Student's t-test of significance was used.

  • b

    Cytogenetics abnormalities identified in AML patients are described in Results.

No. of patients371819 
Patient age (mean)423945NS
No. of male patients1798NS
No. of female patients20911
AML type
WBC (mean /10−9 L; N = 35)291936NS
Peripheral blood monocyte count (mean /10−9 L; N = 35)4720.080
Peripheral blood blasts (mean % of WBC; N = 36)2512390.009
Bone marrow blasts (mean % of nucleated cells; N = 30)524954NS
Hemoglobin (mean, g/dL; N = 35)999NS
Platelets (mean /10−9 L; N = 37)555951NS
Cytogenetic abnormalities (N = 32)b
Adverse prognosis
Favorable prognosis
Intermediate prognosis

Among the cellular markers used for flow cytometric diagnosis of AML, none was as sensitive or specific as ILT3 for distinguishing AML with monocytic differentiation from other forms of AML (Fig. 3). Although CD11c, CD33, and HLA-DR were positive in >90% of the patients with AML M4/M5, these markers lacked specificity as they were also expressed in a significant fraction of patients with other AML types. CD11c and CD33 were positive in >50% and 100%, respectively, of the patients with AML M1/M2 and M3, while HLA-DR was positive in >80% of patients with AML M1/M2. Of note, the monocytic marker CD14 was expressed in only 11 out of 18 cases (61%) of AML displaying monocytic differentiation. CD64, a monocytic marker used for identification of monocytic AML by flow cytometry (32, 33), was expressed in 80% of monocytic AML cases, 67% of AML with minimal differentiation, and 88% of APL cases. Thus, CD64 was much less specific than ILT3 in identifying AML with monocytic differentiation.

Figure 3.

Expression of relevant cell markers analyzed by flow cytometry in 37 patients with acute myeloid leukemia.

In patients with AML displaying monocytic differentiation, ILT3 was expressed by leukemic cells at various stages of maturation. As illustrated in Figure 4 (panels A, B, C), ILT3 was co-expressed by CD34+/- CD117+CD14- monoblasts and promonocytes as well as by more differentiated CD34-CD117-CD14+/- leukemic cells. Overall, co-expression of ILT3 and CD117 was observed in 50%, while co-expression of ILT3 and CD34 was seen in 39% of cases with AML displaying monocytic differentiation (Fig. 3). As shown above, ILT3 was absent on leukemic cells from patients with AML lacking features of monocytic differentiation (Figs. 4E and 4F).

Figure 4.

Flow cytometric analysis of whole bone marrow aspirates obtained from patients with acute myeloid leukemia. Leukemic cells were gated based on abnormal immunophenotypic and light scatter features. The results are representative for: AML with monocytic differentiation (M4/M5—Panels AD), AML without differentiation (M1/M2—Panel E), and APL (M3—Panel F). Cell surface expression of ILT3, CD14, CD34, CD45, and CD117 is depicted. [Color figure can be viewed in the online issue which is available at]

To determine whether ILT3 can be used for post-treatment monitoring of patients with AML displaying monocytic differentiation, we analyzed ILT3 expression in 13 samples obtained from AML patients with relapsed or refractory disease. Seven out of 13 samples were from patients with AML displaying monocytic differentiation (M4/M5), while six were from patients with M1/M2 AML. The leukemic cells present in all seven cases with relapsed or refractory AML M4/M5 displayed surface ILT3, indicating that ILT3 expression was preserved after treatment. This is illustrated in Figure 4D, which shows the flow cytometric results obtained post-treatment from one of the patients previously diagnosed with AML with monocytic differentiation (presented in Fig. 4A). The results presented in Figure 4D were obtained three years after the initial diagnosis and provided evidence of leukemia relapse. In this bone marrow aspirate, the CD45dim/low SSC cells accounted for 4% of all nucleated cells and comprised cells expressing CD4, CD11c, CD33, CD34, CD64, CD117, HLA-DR, and predominantly negative for CD14, a phenotype consistent with that of the original leukemia. Leukemia relapse was confirmed by cytogenetic analysis. As shown in Figure 4D, ILT3+ cells accounted for 87% of the CD45dim/low SSC cells, significantly more than that observed in noninvolved BM (Fig. 2). Thus, ILT3 can be a useful marker to identify residual or relapsed disease in patients with AML with monocytic differentiation. In contrast, the leukemic cells from patients with recurrent M1/M2 AML were ILT3 negative (data not shown). These results indicate that ILT3 may be a useful marker for disease monitoring.

Clinical Correlations

There was a statistically significant correlation between ILT3 expression and the AML type. ILT3 was positive in all AML M4/M5 cases yet in none of the M1/M2 and M3 cases (P < 0.0001; Table 2). Although the frequency of leukemic blasts present in the BM did not vary significantly between the ILT3+ and ILT3- AML groups, the frequency of circulating leukemic blasts was significantly lower in the ILT3+ group compared with the ILT3- group (P < 0.009; Table 2. Monocytosis occurred more frequently in patients with ILT3+ AML compared with patients with ILT3- AML, although the difference did not reach statistical significance (P = 0.08). The association between ILT3 expression and the frequency of circulating leukemic blasts or monocytosis most likely reflects the association between ILT3 expression and monocytic differentiation. There were no significant differences between the two groups for the following parameters: age, gender, WBC, hemoglobin levels, and platelet count.

Cytogenetic data were available for 32 patients with AML. Of these, 27 patients carried chromosomal abnormalities. The following cytogenetic abnormalities associated with unfavorable outcome in patients with AML (11) were detected in our study patients: 5q deletion, monosomies of chromosomes 5 and/or 7, and complex karyotype (>3 unrelated abnormalities). One or more of these abnormalities were detected in 5 out of 17 patients with ILT3- AML (M1/M2 group), yet in none of the patients with ILT3+ AML (P = 0.046; Table 2). The cytogenetic abnormalities associated with a favorable prognosis were: t(15;17), detected in 7 out of 10 patients with AML M3 (APL), and Inv16, detected in one patient from the ILT3+ M4/M5 group. The most frequent abnormalities found in the ILT3+ AML group were those associated with an intermediate prognosis, such as trisomy 8, t(4;11) and t(9;11) (Table 2; P = 0.001). Cytogenetic abnormalities previously associated with monocytic differentiation, such as t(9;11) and 16q22, were observed in only 3 of 14 patients with AML M4/M5. As indicated by multiple regression analysis, the AML type remained the only clinical parameter strongly correlated with ILT3 expression (P < 0.001).


In this study, we show that the inhibitory receptor ILT3 is a highly sensitive and specific marker for the diagnosis and monitoring of AML with monocytic differentiation. ILT3 was expressed by 18/18 cases of AML with monocytic differentiation and in none of the 19 cases of AML, which included M1/M2 and M3 cases. The distinction between monocytic AML and other AML types is extremely important particularly in the differential diagnosis of AML with monocytic differentiation and microgranular APL, two leukemia subtypes which require different treatment strategies (4,10). Although the number of microgranular APL was relatively small (three out of nine APL cases), our data suggest that ILT3 can be used to distinguish AML with monocytic differentiation from microgranular APL. ILT3 may be also a useful marker in the differential diagnosis of cutaneous lesions, such as myeloid sarcoma occurring in patients with monocytic AML versus lymphoma or soft tissue sarcoma (1,15).

The definition of risk categories for patients with AML is largely based on cytogenetic and molecular parameters (5, 6, 8, 11). However, new biomarkers are greatly needed, particularly in patients with cytogenetically normal AML. In our study, the most frequent cytogenetic abnormalities observed in patients with ILT3+ AML were those associated with intermediate prognosis and included trisomy 8, t(4;11) and t(9;11) (11). A limitation of our study is the lack of data on molecular markers for AML (5,6). Thus, the potential value of ILT3 as a prognostic marker, particularly in cytogenetically normal AML, remains to be determined.

Detection of minimal residual disease (MRD) has been recognized as a powerful prognostic indicator in patients with acute lymphoblastic leukemia (34). Emerging evidence indicates that MRD detection in patients with AML is also associated with poor prognosis, and early therapeutic interventions may be of clinical benefit (35,36). Although the presence of rare ILT3 positive immature cells in noninvolved bone marrow may pose some difficulties, our results suggest that ILT3 is a candidate marker for MRD detection in patients with AML displaying monocytic differentiation due to its high sensitivity, specificity, and stable expression.

Co-expression of ILT3 and the early markers CD34 and CD117 by myeloid leukemic blasts may be interpreted as a maturation asynchronism of the abnormal cells. However, rare precursor cells carrying the ILT3+CD34+CD117+ phenotype were also identified in noninvolved BM. Thus, this phenotype may represent a normal feature of early precursors committed to the monocytic lineage. Current models propose that monocytes, certain macrophage subsets, and myeloid dendritic cells originate from hematopoietic stem cell-derived progenitors with myeloid restricted differentiation potential (37). However, it is not clear whether these progenitors can only differentiate into mononucleated cells or can also give rise to polymorphonuclear cells. This question is difficult to address since early monocytic/granulocytic precursors cannot be discriminated based on their phenotype. Given that ILT3 is expressed by monocytes but not by granulocytes, we can speculate that ILT3 expression acquired by early progenitors, as shown in our study, marks the commitment to monocytic lineage. Thus, our findings favor the hypothesis that segregation of myeloid and monocytic precursors occurs at an early step of hematopoietic differentiation (38).

The inhibitory effect of ILT3 on T cell activation has been clearly demonstrated (26–28). Although the ILT3 ligand is not yet known, the finding that a soluble form of ILT3 can directly suppress T lymphocyte function argues for the existence of a functional ligand on the surface of T cells (39,40). Expression of ILT3 by leukemic cells, as found in our study, may contribute to the inhibition of tumor specific T cell responses. It has been previously shown that transfection of the ILT3 gene in KG-1 cells, a human acute myelogeneous cell line, resulted in significant reduction of allogeneic T cell responses in vitro and in vivo (26). These findings, in conjunction with our current results, highlight the potential value of ILT3 as a target for therapy in AML with monocytic differentiation. Blocking ILT3 signaling with specific antibodies or antagonists may render ILT3+ AML cells more susceptible to differentiation agents and anti-tumor T cell responses (29).

In conclusion, ILT3 is a highly sensitive and specific marker which distinguishes AML with monocytic differentiation from other types of AML. Testing of ILT3 expression should be incorporated both into the initial diagnostic work-up and monitoring of patients with AML.