Dysfunction of immune system in the development of large granular lymphocyte leukemia

ABSTRACT

Objectives: Large granular lymphocyte (LGL) leukemia is a rare type of lymphoproliferative disease caused by clonal antigenic stimulation of T cells and natural killer (NK) cells.

Methods: In this review, we focus on the current knowledge of the immunological dysfunctions associated with LGL leukemia and the associated disorders coexistent with this disease. Novel therapeutic options targeting known molecular mechanisms are also discussed.

Results and Discussion: The pathogenesis of LGL leukemia involves the accumulation of gene mutations, dysregulated signaling pathways and immunological dysfunction. Mounting evidence indicated that dysregulated survival signaling pathways may be responsible for the immunological dysfunction in LGL leukemia including decreased numbers of neutrophils, dysregulated signal transduction of NK cells, abnormal B-cells, aberrant CD8+ T cells, as well as autoimmune and hematological abnormalities.

Conclusion: A better understanding of the immune dysregulation triggered by LGL leukemia will be beneficial to explore the pathogenesis and potential therapeutic targets for this disease.

KEYWORDS:

• Large granular lymphocyte leukemia
• neutropenia
• hematological abnormality
• targeted therapy

Introduction

Large granular lymphocyte (LGL) leukemia is identified as a rare type of lymphoproliferative disorder that results from the clonal expansion of large granular lymphocytes (LGLs) [1–6]. As key innate immune effectors, LGLs occupy roughly 10–15% of the peripheral blood mononuclear cells (PBMCs) and originate from two main lineages, CD3+ cytotoxic T-lymphocytes (CTLs) and CD3- natural killer (NK) cells [7–10]. In 2008, a provisional entity of chronic NK-cell lymphoproliferative disorder (CLPD-NK) was recognized by the WHO in order to distinguish it from the aggressive NK-cell leukemia. Thus there are three kinds of LGL disorders, namely T-cell large granular lymphocyte (T-LGL) leukemia, aggressive NK-cell leukemia and CLPD-NK [1,11,12]. The latest 2016 WHO guidelines followed the previous classification method, but specially underlined the detection of STAT mutations as a new subtype [11,13]. In fact, LGL leukemia can be considered as a result of the clonal expansion of either NK cells or T cells [2]. The abnormal proliferation of LGLs contributes to the development and progression of the immune system disorders in humans [8], including the autoimmune diseases, hematological abnormalities, and dysregulated signal transduction of T cells and NK cells, besides the B-cell dyscrasias. Consequently, LGL leukemia is an excellent model for the study of hematological cancers that manifest immune system dysregulations. Herein we mainly focus on the impaired homeostasis of the immune system in LGL leukemia and briefly introduce the novel therapeutic agents targeting the corresponding abnormalities in LGL leukemia.

Main text

1. Abnormal neutropenia in LGL leukemia patients

Neutropenia, the most common cytopenia, occurring in 80% of patients attacked by indolent T-LGL leukemia and in a lesser degree of NK-LGL leukemia, is identified as the reason for recurrent bacterial infections [8,10,14]. Although the mechanism of neutropenia is not fully understood [7], the acquired neutropenia in LGL leukemia has been intensively studied. It results from the inadequate neutrophil production and incremental neutrophil destruction [10]. Unlike congenital neutropenia delineated elsewhere, the examination of the bone marrow in T-LGL leukemia patients exhibits mild hypercellularity and left-shifted myeloid maturation [15]. The atypical production defects observed imply a significant role of proliferation and survival defects in the development of neutropenia [16]. The presence of anti-neutrophil antibodies and high levels of soluble Fas ligand in LGL leukemia, respectively, suggested the antibody-mediated neutrophil destruction and receptor-mediated target apoptosis in the peripheral blood. In addition, clonal expansion, bone marrow infiltration and abnormal immune activity of leukemic LGLs also play an essential role in neutrophil deficiency observed in LGL leukemia patients [7,8,17]. Moreover, these mechanisms are not mutually exclusive but interact with each other in a patient with T-LGL [16]. Recently, a case report of an acute promyelocytic leukemia patient with chronic severe neutropenia and related LGL expansion also suggested the multifactorial origin of neutropenia and that immune activation/dysregulation may be conducive to the progression of neutropenia [18].

Indications for treatment of T-LGL leukemia contain severe neutropenia (< 500/mL) or recurrent bacterial infections caused by less severe but chronic neutropenia [16]. Immunosuppressive therapies like the application of low-dose methotrexate (MTX), cyclophosphamide, or cyclosporine slowly and effectively alleviate neutropenia while the effect of using granulocyte-macrophage-colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) alone is usually partial and transient [10,19]. So, the hematopoietic growth factors may be more useful for rapid neutropenia correction in febrile neutropenia patients and conditions in combination with other agents [20–22]. Splenectomy was not recommended as a first-line therapy owing to the lack of evidence for the splenic destruction of neutrophils in LGL leukemia and insufficient data on its efficacy in treating the LGL leukemia-related neutropenia [10,19]. A retrospective report of 15 splenectomized patients revealed a hematological response and remission of cytopenia in all patients [23], which indicated that splenectomy may be more effective in severe splenomegaly or evidential cytopenia [19]. Alternatively, CD52 has been reported to express on abnormal cells in T-LGL leukemia and a study of eight patients with refractory cytopenia treated using Alemtuzumab (anti-CD52 mAb) showed remission of neutropenia in one of three patients [24].

2. Activation of NK cells and its signal transduction in LGL leukemia

‘LGL leukemia’ was termed based on observation of clonality and certification of tissue invasion by LGL of marrow, spleen, liver, and lung [8–10,14,25,26]. NK-LGL leukemia may have a chronic or acute course [27,28]. Features of aggressive NK-LGL leukemia include high numbers of circulating NK cells, hepatosplenomegaly, and systemic symptoms [27]. Chronic NK-LGL leukemia follows a more indolent course and is commonly associated with anemia, neutropenia, rheumatoid arthritis, and occasionally idiopathic primary pulmonary hypertension (PPH) [28]. Infiltrating LGLs are associated with endothelial damage and increasing lung fibrosis in patients with LGL leukemia [29]. Unfortunately, the etiology triggering the activation of NK cells is not fully elucidated. It has been hypothesized that infection with EBV (Epstein–Barr virus) or HTLV (Human T-cell leukemia/lymphoma virus)-like virus may be an initial trigger for the activation of NK clones in such patients [30,31]. Nevertheless, it is confirmed that the infiltrating leukemic cells have an association with direct tissue destruction in LGL leukemia and activated NK cells may play a pivotal role in this process [25].

Fischer et al. reported an altered expression of NK-associated markers in all the LGL lymphoproliferation (LGLL) patients (n = 13) enrolled in their study. Killer-immunoglobulin-like receptors (KIR) were either overexpressed in some cases or were lacking in others. While CD94, NKG2A, and CD85j were overexpressed, the expression levels of NKp30 and NKp46 were distinctly decreased in NK-LGLL. These novel NK markers may serve as pathological features of LGLL and facilitate the diagnosis of LGL proliferation [32]. A Ras-independent pathway (PI3K→ Rac1→ PAK1→ MEK→ MAPK/ERK) triggers lytic function by granule mobilization in NK cells [33,34]. Burnette and Wei demonstrated the in vitro activation of NKR signaling through DAP10 and DAP12 adaptor proteins mediate the lysis of pulmonary endothelial cell line by the NK cells isolated from the NK-LGL leukemia patients. Targeted disruption of DAP10 and DAP12 will be therefore beneficial for alleviating the tissue destruction in LGL leukemia or other autoimmune diseases [35]. A Ras-dependent pathway, Ras-MEK1-ERK, is constitutively activated in NK-LGL leukemia [32]. Activation of Ras controls the diverse extracellular signals involved in proliferation, survival, and differentiation of the cells [33], and its inhibition induces apoptosis in leukemic LGLs [13]. Furthermore, in view of the suppression of the Fas-mediated signaling by the MEK/ERK pathway, the inhibition of this pathway may restore Fas sensitivity in leukemic LGLs [32]. The drug Tipifarnib (also known as R1150777, Zarnestra) is a Ras farnesyltransferase inhibitor specially designed to inhibit the Ras pathway [34]. Tipifarnib was efficient in relieving the symptoms of pulmonary hypertension in an NK-LGL leukemia patient suffering from the primary pulmonary artery hypertension (PAH) [35]. However, an NCI phase II clinical trial with Tipifarnib (#NCT00360776) involving eight patients failed to show any promising clinical hematologic response [1,13,34].

3. B-cell dysfunction in LGL leukemia and related diseases

There is an enhanced immunoglobulin (Ig) production in half the patients developing LGL/natural killer cell proliferative disease (LGL/NK-PD). Other altered B-cell activities, such as hypergammaglobulinemia, the presence of rheumatoid factor, anti-nuclear or anti-neutrophil antibodies have also been observed [36–38]. This phenomenon may partly be due to the incapacity of abnormally expanded LGLs to restrain the production of immunoglobulins in vivo [36]. The similar non-incidental occurrence of B-cell dyscrasias in the T-LGL leukemia has been described in numerous literatures, including monoclonal gammopathy of unknown significance (MGUS) [39], Hodgkin’s disease [40], chronic lymphocytic leukemia (CLL) [41], hairy cell leukemia [42], marginal zone lymphoma [1], lymphoplasmacytic lymphoma [43], with MGUS being the most common LGL leukemia related disorder [39]. Although the simultaneous occurrence of T-LGL leukemia and B-cell dyscrasias may not be fortuitous, the association between the two diseases is not fully clarified [39,43]. It is worth noting that several hypotheses explaining this phenomenon have been proposed. One of the hypothesis proposes that both the T- and B-cell clonal proliferations originate from a common leukemic stem cell [44], while other and more reasonable explanations include the theory that the clonal expansion of both B and T cells is caused by the clonal antigenic stimulation resulting in the co-occurrence of T-LGL leukemia and B-cell dysfunction [45], and the idea that the B-cell dysfunction may fail to completely eliminate the inciting pathogen, resulting in the clonal T-cell expansion triggered by the chronic antigenic stimulation and to the consequent T-LGL leukemia [39]. Additionally, there is a prevalent theory that the dysregulated immune system in LGL leukemia contributes to the occurrence of these B-cell disorders mentioned above [38]. Overall, these hypotheses collectively emphasize the importance of the dysregulated immune system in LGL leukemia and clonal B-cell disorders. This allows us to speculate that the two diseases may interact with and promote each other in the dysregulated immune microenvironment. Nevertheless, further studies are required to provide insights into the accurate pathophysiological mechanism behind the coexistence of LGL leukemia and B-cell dysfunction.

CD52 is expressed in T and B lymphocytes as well as monocytes under normal conditions, and the observed high expression levels of CD52 in Leukemic LGLs provides a therapeutic rationale for use in T-cell LGL leukemia patients [1,60]. Alemtuzumab has been proved to be efficient in treating various chronic B- and T-cell malignancies, such as T-cell prolymphocytic leukemia, chronic lymphocytic leukemia (CLL) as well as MDS [1]. In a recent prospective phase II nonrandomized single arm study (#NCT00345345), 56% (14) patients achieved a hematological response in a cohort of 25 patients with T-LGL leukemia. Nevertheless, the infection risks limit the availability and clinical application of the agent [1]. RituximAb, a mAb specially targeted for CD20, a B-cell membrane protein, is useful in treating T-LGL leukemia coexisting with RA and acquires a long-time remission [26]. This unexpected outcome of the specific anti-B-cell agent in such a monoclonal T-cell disease makes LGL leukemia seem like a reactive response to chronic antigen stimulation instead of true malignancies.

4. Abnormal CD8+ T cells in LGL leukemia

T-LGL leukemia presents as a CD3 + CD8 + CD57 + TCRαβ+ phenotype in most cases, which signifies a constitutively activated phenotype of T cells [11,38,46]. There is a significant accumulation of CD8+ terminal effector memory T cells characterized by CD3+ CD8+ CD45RA+ CD62L− phenotype in the patients in some cases of T-LGL leukemia [47,48]. T-LGL leukemia can be deemed as the polarized expansion of these CD8+ T cells [48,49]. The molecular analysis of clonal T-cell receptor (TCR) repertoire in LGL leukemia is therefore essential for the identification of clonal expansion and the detection of clinical response [50]. The increased number of CD8+ T cells can mediate normal tissue destruction in LGL leukemia [25]. Actually, the autoimmune diseases, especially rheumatoid arthritis (RA) and pulmonary arterial hypertension (PAH), are often regarded as a significant manifestation of the LGL leukemia [10,25,51]. Similar to the dysregulated NK signaling pathway mentioned above, Wei et al. observed that CD8+ T cells from LGL leukemia patients present high levels of multiple NKRs as well as their adapter proteins DAP10 and DAP12, and lead to the constitutive activation of multiple signaling intermediates within the NKR/DAP10/DAP12 signaling cascade [25]. This discovery may account for the excessive normal tissue destruction in LGL leukemia and other autoimmune diseases [25].

Interestingly, the malignant amplification of T cells is a combined effect of numerous factors and involves the activation of multiple pathways. One of the major recognized mechanism is the dysregulation of Fas-FasL-mediated apoptosis [13,14,49]. Fas-mediated apoptosis is not only a major mechanism for the cytotoxic T cells (including LGLs) mediated induction of cell death in infected or foreign cells [14,51,52], but it also exerts an important impact on the homeostasis of T cells through the elimination of excessive antigen-activated effector lymphocytes [10,14,47,48]. Although high levels of Fas and FasL are expressed on the surface of leukemic LGLs, these cells exhibit resistance to Fas-mediated apoptosis [10,47,48,52]. Compared to the healthy controls, the observed elevated level of soluble form of Fas (sFas) in the sera of LGL leukemia patients may serve as a decoy receptor instead of Fas and thus mediate the apoptotic resistance in leukemic LGLs [47]. Additionally, the NF-κB signaling pathway is also dysregulated in LGL leukemia [1,49]. NF-κB is a transcription factor complex involved in the regulation of inflammation, hematopoiesis as well as the proliferation and survival of immune cells [53]. A member of NF-κB family, c-Rel, is overexpressed in LGL leukemia [54] and NF-κB is constitutively activated in T-LGLs [52]. The activation of NF-κB promotes the growth and function of T cells, while its inhibition causes AICD (activation-induced cell death) or apoptosis of T cells [53]. Besides, the cytokines and growth factors are critical for the growth and proliferation of T cells. IL-15 plays an important role in facilitating the survival and proliferation of T and NK cells [55], and PDGF acts downstream through many survival signaling pathways such as JAK-STAT, Ras-MEK1-ERK, and PI3K-Akt [56]. A model experimentally verified that the initial activation of T cells, constitutive release of IL-15, and activation of PDGF signaling were sufficient to generate all the known dysregulations reported in leukemic T-LGLs [52].

The matrix metalloproteinase (MMP) inhibitors may be applied in the treatment of LGL leukemia. A MMP-like enzyme can transform human FasL into a soluble form of FasL (sFasL) through alternative splicing or proteolytic cleavage [57]. Therefore, MMP inhibitors could theoretically prevent the generation of soluble FasL and lower its levels in sera. Bortezomib, a proteasome inhibitor down-regulating the NF-kB pathway, is considered to be a promising therapeutic agent in the treatment of LGL leukemia [58]. It was already permitted by the FDA in the treatment of mantle cell lymphoma and relapsed multiple myeloma [59]. In vitro studies have reported the efficiency of bortezomib in inducing the apoptosis of leukemic LGLs, nevertheless, the clinical trials with this agent are required to prove its real time efficacy against LGL leukemia [1]. Considering the essential role of IL-15 in the survival of T cells, a monoclonal antibody, Mikβ1, targeting CD122 (β-subunit of IL-2 and IL-15 receptors) has been tested in two phase I clinical trials and no toxicity and immunogenicity was reported to the antibody [60,61]. Although no significant clinical response was observed in both the trials, 58% (7/12) of the patients in one of the trials demonstrated a lowered expression of CD122 on the leukemic cells [60]. As mentioned above, these agents may be reasonable and promising therapeutic choices, although further clinical trials are demanded to prove their safety and efficacy.

5. Autoimmune disorders and hematological abnormalities related to LGL leukemia

LGL leukemia can be deemed as an example of hematological cancer with the dysregulated immune system, in particular, T-cell leukemia [17,51,62]. The most common hematological disorder is neutropenia, which is observed in 70–80% T-cell LGL leukemia patients. Other disorders such as anemia and thrombocytopenia, hyper- and hypo-gammaglobulinemia, hereditary hemochromatosis, splenic marginal zone lymphoma, peripheral T lymphoma and Wiskott–Aldrich syndrome (WAS) also accompany LGL leukemia, nonetheless, with a lower incidence [7,10,38]. These disorders are closely associated with the elevated B-cell activity described above. In addition, other hematological problems especially bone marrow failure syndromes can also occur in both T-and NK-LGL leukemia, involving aplastic anemia (AA) [63–65], hemolytic anemia (HA) [66–68], paroxysmal nocturnal hemoglobinuria (PNH) [39,64,69], and myelodysplastic syndrome (MDS) [64,70,71]. The co-occurrence of these hematological disorders with LGL leukemia is very common, several paradigms may throw light on the underlying pathologic and immunogenic mechanisms. In MDS, it has been indicated that abnormal marrow progenitors initiate the incipient clonal expansion of LGL that exert immunopathological damage to the circulating hematologic cells and lead to a deficient proliferation state as neutropenia, thrombocytopenia, AA (Aplastic anemia), and PRCA (pure red cell aplasia) [10,72]. However, in WAS (a rare X-linked immunodeficiency disease generated by WASP gene mutation), gene mutation seems to create a favorable environment for clonal LGL growth [45,73–75].

The LGL leukemia can be accompanied by a spectrum of autoimmune disorders, such as RA (the most familiar autoimmune disease correlated with T-LGL leukemia) [39,76–79], Felty’s syndrome (analogous to the combination of neutropenia, RA, and splenomegaly) [78], PAH (pulmonary artery hypertension) [35,80], autoimmune thyroiditis (Hashimoto’s disease) [23,79,81], autoimmune polyglandular syndrome (APS) [82], Grave’s disease (hyperparathyroidism), and Cushing’s syndrome [6,23,79]. Felty’s syndrome and T-LGL leukemia with RA appear to be a part of same disease process since the human leukocyte antigen DR4 (HLA-DR4), an immunogenic marker, is prevalent in both the diseases [6,10,12,14,78]. Besides, the remarkable similarities of substantial clinical overlap and the genetic data between them implied that they were not separate entities but a rather continuous spectrum of the same disorder [6,10,12,83,84]. In addition, there are reports highlighting that the LGL leukemia and RA share a common pathogenesis encompassing the protein homologs of BA21 [85–89]. As mentioned above, a case report showed a long-term remission of T-LGL leukemia with RA after RituximAb therapy in two patients [26]. RituximAb is a monoclonal anti-CD20 antibody [90–92], and it has been approved for treating RA on account of inducing transient B-cell depletion [90]. Nevertheless, the long-term remission of T-LGL leukemia is probably attributed to the change of immune microenvironment, especially affecting cytokine balance competent of vitiating the LGL clone survival [26]. In general (Table 1), all these studies revealed the dysregulated immune function associated with LGL leukemia, although its detailed molecular mechanisms need to be further investigated in order to develop a therapeutic strategy to restore the immune homeostasis in LGL leukemia patients.

Table 1. Paradigms correlated with LGL leukemia.

Disease	Mechanism	Reference
MDS	Abnormal marrow progenitors trigger incipient clonal expansion of LGL and finally leukemia LGLs lead to hematological abnormalities in LGL leukemia	[10,29]
WAS	Gene mutation seems to play a vital role in LGL expansion	[45,71–73]
Felty’s syndrome	Felty’s syndrome and T-LGL leukemia with RA may be a continuous spectrum of the same disorder	[6,10,12,15,76,82,83]
RA	A common pathogenesis was recognized in LGL leukemia and RA	[84–88]

6. Therapeutic interventions for the disordered immunity in LGL leukemia

Immunosuppressive therapy is the basis for the treatment of LGL leukemia since the LGL leukemia originates from the clonal expansion of constitutively activated cytotoxic lymphocytes [11]. The first-line therapy depends on the immunosuppressive drugs incorporating MTX, cyclophosphamide and Cyclosporine A [1,14,93,94]. Nevertheless, the exploration and experimentation for the discovery of novel potential treatment alternatives has never stopped (Table 2). Monoclonal antibodies targeting specific receptors expressed on the leukemic LGLs and molecular inhibitors targeting survival signaling cascades activated in the tumor cells seem potential candidates for the treatment of LGL leukemia. Besides the Alemtuzumab (anti-CD52 mAb) and Mikβ1 (anti-122 mAb) described above, a phase I trial using SiplizumAb (anti-CD2 mAb) was conducted in patients with adult T-cell leukemia/lymphoma (ATLL) including T-LGL leukemia. Unfortunately, four of the patients developed a severe Epstein–Barr virus (EBV)-driven lymphoproliferative disorder and the trial has been halted. The JAK-STAT3, PI3K-Akt, sphingolipid rheostat, and other pathways can also be targeted apart from Ras-MEK1-ERK, NF-κB, and IL-15 signaling pathways mentioned above. Several reports mention that these pathways are intricate and interactive rather than being mutually independent thus result in the imbalance of the hematological system [1,11,13,14,52,93]. Recently, a gene sequencing analysis revealed that 40% of the LGL leukemia patients carried mutations in the SH2 domain of STAT3 and were more likely to develop neutropenia and RA [95]. Furthermore, a multicenter phase II study using methotrexate in patients with the Y640F mutation in STAT3 showed a promising response to the treatment of LGL leukemia [14]. It can be concluded that the gene mutations contribute equally to the dysregulated survival pathways and thus could be the potential therapeutic targets for the treatment of LGL leukemia.

Table 2. Novel potential treatment options for LGL leukemia.

Category	Agent	Mechanism	Application	Reference
mAb	Alemtuzumab	Targets CD52 on T and NK cells	chronic B- and T-cell malignancies	[1,46]
mAb	Mikβ1	Targets CD122	Waited further investigation	[60,61]
mAb	RituximAb	Targets CD20 on B cells,	LGL leukemia with RA	[11,17]
MMP inhibitor	Marimastat	Inhibits the conversion from FasL to sFasL	Waited further investigation	[10,48,49,52,57]
Ras farnesyltransferase inhibitor	Tipifarnib	Inhibits ERK activity	LGL leukemia with PAH	[13,33–35]
Pan-PI3 K inhibitors	PX-866, GDC-0941	Targets PI3K–Akt signaling pathway	Waited further investigation for various side effects	[10,11,13]
Pathway inhibitors	OPB-31121 (STAT3 inhibitor) CP690550 (Jak3 inhibitor)	Targets JAK-STAT3 pathway	Hematopoietic malignancies (OPB-31121), refractory LGL leukemia and RA (CP690550)	[96–98]
S1PR agonist	FTY720	Targets sphingolipid rheostat pathway	A promising agent in the treatment of NK-LGL leukemia	[99]
Protease inhibitor	Bortezomib	Down-regulates NF-κB signaling pathway	Waited further investigation for insufficient clinical trials	[100,101]

Conclusions

The abnormal clonal proliferation of LGL tightly correlates with the disease progression and impaired homeostasis of the immune system in vivo. Hematological and autoimmune disorders are often observed in LGL leukemia patients associated with neutropenia. Besides, the coexistence of activated NK cells is discovered in LGL leukemia, resulting in an altered expression of NK-associated markers, activated NKR-DAP10/DAP12 signaling cascade and constitutively activated anti-apoptotic Ras-MEK1-ERK pathway. Inhibition of DAP10 and DAP12 can reduce the CD8+ T-cell mediated tissue damage, suggesting their potential to be used as therapeutic targets for this disease. Other dysregulated survival signaling pathways detected in LGL leukemia like Fas-FasL, Ras-MEK1-ERK, NF-κB and IL-15 signaling pathways can also provide useful targets for exploring more effective and accurate drugs against LGL leukemia. A highly frequent B-cell abnormality also coexists in T-LGL leukemia and results in a range of hematological abnormalities with the most common being the MGUS. These immunological observations may provide insights into the pathophysiological mechanisms involved in T-LGL leukemia. Novel insights into the dysregulated signaling pathways and immune system abnormalities will assist in the identification of more beneficial therapeutic molecular targets, to find better and more specific therapeutic methods to treat LGL leukemia.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by a grant from National Science and Technology Supporting Program (No.2015BAI12B12), National Natural Science Foundation of China (No. 81572265 and No. 31500736) and National Key R&D Program (2018YFC1313400).