key: cord-0730990-sq0ob4m9
authors: Vitale, Candida; Boccellato, Elia; Comba, Lorenzo; Jones, Rebecca; Perutelli, Francesca; Griggio, Valentina; Coscia, Marta
title: Impact of Immune Parameters and Immune Dysfunctions on the Prognosis of Patients with Chronic Lymphocytic Leukemia
date: 2021-07-30
journal: Cancers (Basel)
DOI: 10.3390/cancers13153856
sha: 36cfaa02a9b7f402fdabb383153506ecddf00bde
doc_id: 730990
cord_uid: sq0ob4m9

SIMPLE SUMMARY: In chronic lymphocytic leukemia (CLL), immune alterations—affecting both the innate and adaptive immunity—are very common. As a clinical consequence, patients with CLL frequently present with autoimmune phenomena, increased risk of infections and second malignancies. The aim of this review article is to present available data on CLL-associated alterations of immune parameters that correlate with known prognostic markers and with clinical outcome. Also, data on the impact of immune-related clinical manifestations on the prognosis of patients with CLL will be discussed. ABSTRACT: Chronic lymphocytic leukemia (CLL) is characterized by a wide spectrum of immune alterations, affecting both the innate and adaptive immunity. These immune dysfunctions strongly impact the immune surveillance, facilitate tumor progression and eventually affect the disease course. Quantitative and functional alterations involving conventional T cells, γδ T cells, regulatory T cells, NK and NKT cells, and myeloid cells, together with hypogammaglobulinemia, aberrations in the complement pathways and altered cytokine signature have been reported in patients with CLL. Some of these immune parameters have been shown to associate with other CLL-related characteristics with a known prognostic relevance or to correlate with disease prognosis. Also, in CLL, the complex immune response dysfunctions eventually translate in clinical manifestations, including autoimmune phenomena, increased risk of infections and second malignancies. These clinical issues are overall the most common complications that affect the course and management of CLL, and they also may impact overall disease prognosis.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease typically characterized by a wide spectrum of immune alterations, affecting both innate and adaptive immunity (reviewed in [1] ). In addition to being a hallmark of the disease, CLL-associated immune dysfunctions strongly impact the immune surveillance, facilitate tumor progression and eventually affect the disease course.

The aim of this review article is to present available data on immune parameters which are altered in patients with CLL, and that can associate with other disease characteristics with a known prognostic relevance or that directly correlate with prognosis ( Figure 1 ). In addition, we will discuss major immune-related clinical manifestations -such as autoimmunity, 

T lymphocytes play a pivotal role in tumor immune-surveillance and immune response to infections. CD4+ T helper lymphocytes are the main coordinator of immune response via both cell-to-cell interactions and cytokine production, activating B lymphocytes and CD8+ cytotoxic T lymphocytes [reviewed in [2, 3] ]. In patients affected by CLL, CD4+ and CD8+ T lymphocyte numbers are increased [4] [5] [6] [7] , and several studies have explored a possible prognostic impact of conventional T-cell counts in this disease. The ratio of T cells:malignant monoclonal B cells (MBC) has been described as an independent predictor of time-to-first treatment (TTFT) in early stage CLL, with higher CD4:MBC and CD8:MBC ratios predicting longer overall survival (OS) [8] . Also, an indolent clinical course (i.e., a probability of OS at 10 years of 95%) has been described in a subgroup of patients characterized by elevated CD8:MBC ratio and the absence of CD38 expression on tumor cells [9] .

In CLL, T cells undergo oligoclonal proliferation that is probably due to the chronic exposure to malignant B cells [10] . The expansion of CD8+ T lymphocytes outweighs that of CD4+, resulting in a reduced (also called inverted) CD4:CD8 ratio [5] [6] [7] 11] , which has been associated with advanced disease stages [6] . Interestingly, Apostolopoulos and colleagues showed that a lower CD4:CD8 ratio is also predictive of recurrent respiratory infections [12] . Consistently, several studies found that an inverted CD4:CD8 ratio is associated with shorter TTFT [7, 13] , progression-free survival (PFS) [14, 15] and OS [13] [14] [15] . In the post-treatment setting, instead, it has been shown that following fludarabinecyclophosphamide-rituximab (FCR) treatment, a higher CD4+ − but not CD8+ − Tlymphocyte count associate with a shorter PFS in patients maintaining a detectable minimal residual disease [16] . In this study, a thorough characterization of the CD4+ Figure 1 . Summary of the main alterations characterizing immune cells of patients with early and advanced stage chronic lymphocytic leukemia. ↓ decreased; ↑ increased; ↑↑ particularly increased.

2.1. T Cells 2.1.1. Conventional T Cells T lymphocytes play a pivotal role in tumor immune-surveillance and immune response to infections. CD4+ T helper lymphocytes are the main coordinator of immune response via both cell-to-cell interactions and cytokine production, activating B lymphocytes and CD8+ cytotoxic T lymphocytes [reviewed in [2, 3] ]. In patients affected by CLL, CD4+ and CD8+ T lymphocyte numbers are increased [4] [5] [6] [7] , and several studies have explored a possible prognostic impact of conventional T-cell counts in this disease. The ratio of T cells:malignant monoclonal B cells (MBC) has been described as an independent predictor of time-to-first treatment (TTFT) in early stage CLL, with higher CD4:MBC and CD8:MBC ratios predicting longer overall survival (OS) [8] . Also, an indolent clinical course (i.e., a probability of OS at 10 years of 95%) has been described in a subgroup of patients characterized by elevated CD8:MBC ratio and the absence of CD38 expression on tumor cells [9] .

In CLL, T cells undergo oligoclonal proliferation that is probably due to the chronic exposure to malignant B cells [10] . The expansion of CD8+ T lymphocytes outweighs that of CD4+, resulting in a reduced (also called inverted) CD4:CD8 ratio [5] [6] [7] 11] , which has been associated with advanced disease stages [6] . Interestingly, Apostolopoulos and colleagues showed that a lower CD4:CD8 ratio is also predictive of recurrent respiratory infections [12] . Consistently, several studies found that an inverted CD4:CD8 ratio is associated with shorter TTFT [7, 13] , progression-free survival (PFS) [14, 15] and OS [13] [14] [15] . In the posttreatment setting, instead, it has been shown that following fludarabine-cyclophosphamiderituximab (FCR) treatment, a higher CD4+ − but not CD8+ − T-lymphocyte count associate with a shorter PFS in patients maintaining a detectable minimal residual disease [16] . In this study, a thorough characterization of the CD4+ population revealed that it mostly consists of regulatory T cells (Tregs, CD25+CD127-FoxP3+), which are known to facilitate relapse and progression in CLL (see below).

The composition of T-cell differentiation subsets and Th1/Th2 cell distribution has also been extensively studied in patients with CLL. A reduction in naïve T cells and an increase in effector T lymphocytes and terminally differentiated memory T lymphocytes were consistently described [10, 14, 17, 18] . Furthermore, most reports agree on the increase of Th1 lymphocytes in patients with CLL compared to healthy controls, whereas regarding Th2 cells the data are controversial [7, 17, [19] [20] [21] . Nevertheless, to our knowledge, to date there are no available data that clearly associate Th1 and/or Th2 cell subset expansion with CLLrelated prognostic factors. Interestingly, Puzzolo and colleagues recently demonstrated that the treatment with the BTK ibrutinib induces a decrease in the Th1/Th2 cell ratio, which is more prominent in patients with unmutated immunoglobulin heavy chain variable region genes (IGHV) status and in those who achieve a complete response to therapy [22] .

Th17 cells represent a subset of pro-inflammatory cells involved in inflammation and autoimmunity, which may play a dichotomous role in cancer [23] . Compared to healthy controls, patients affected by CLL have an increased frequency and absolute count of Th17 lymphocytes [17, 21, 24, 25] . Concerning their role on disease evolution, a higher Th17-cell number has been correlated with early stages of the disease [26] , and most [25, 27, 28] -but not all [26] -available data have reported a positive impact of high Th17-cell counts on OS.

In CLL, as in many other cancers, the tumor-mediated chronic antigenic stimulation of T cells also determines an increased surface expression of typical markers of activation [29, 30] , and affects their phenotypic and functional features. Leukemic B cells, through a direct cell-to-cell contact, modify the expression of genes involved in CD4+ and CD8+ T-cell differentiation and function [31] , confirming the role of the malignant cells in nourishing a favorable pro-tumoral microenvironment. Several studies have reported that T lymphocytes from patients with CLL are primed for anergy because of their higher expression of immune-checkpoint molecules, such as CTLA-4 [32] , PD-1 [18, 33] , LAG3 [34] , Tim-3 [35] , TIGIT [36] , CD160 and CD244 [37] , leading to a microenvironment characterized by reduced T-cell proliferation, killing ability and cytokine release. Of note, both CD8+ and CD4+ T-cell subsets show a complex co-expression of molecules indicating T-cell exhaustion (PD-1), senescence (KLRG-1) and activation (HLA-DR) [15] .

Despite T-cell phenotypic and functional alterations have been extensively described in CLL, only few parameters have been reported to have a prognostic relevance. For example, higher soluble LAG3 levels in patients' sera were found to be associated with progressive disease status and shorter TTFT [34] . These findings are possibly attributable to the higher expression of LAG3 mRNA detected in CLL cells with unmutated IGHV compared to mutated IGVH and normal B cells. Also, PD-1 upregulation was described in both CD4+ and CD8+ T cell subsets [18] , and was associated with disease progression [14, 15, 17] and shorter TTFT [14] . Consistently, in a cohort of 80 patients with CLL, Palma et al. found that those with progressive disease had higher CD4+PD1+ and CD8+PD1+ T cells compared to both healthy controls and to non-progressive patients, with a bigger difference noted in previously treated patients [17] . Moreover, among previously treated patients, those with advanced disease stages showed an increased PD1+, Tim-3+ and TIGIT+ T-cell counts [35] . In line with these data, Jimenez et al. recently reported that patients who are in clinical progression present an accumulation of terminally exhausted effector CD8+ T cells, which are characterized by the co-expression of PD-1, CD244 and CD160 inhibitory receptors and by an altered gene expression profile [38] . Interestingly, these specific CD8+ T-cells alterations are possibly mediated by IL-10 released by leukemic cells [38] . Finally, Elston and colleagues examined in their cohort of 74 patients whether specific T-cell subsets could be associated with inferior clinical prognosis [15] . In multivariate analysis, higher percentages of circulating CD4+HLA-DR+PD1+ T-lymphocytes were associated with a shorter PFS.

γδ T lymphocytes are a subset of non-MHC-restricted T lymphocytes which account for 2 to 10% of circulating T cells in healthy subjects. γδ T cells have the ability to kill their targets via cytotoxic mechanisms and, thus, are involved in infection responses, autoimmunity and tumor immune surveillance [39] .

The main circulating subset of γδ T lymphocytes is represented by Vγ9Vδ2 T cells, which recognize non-peptidic phosphoantigens, such as isopentenyl pyrophosphate and aminobisphosphonates, in a T cell receptor (TCR)-dependent fashion [40, 41] . Vγ9Vδ2 T cells collected from patients with CLL show dysfunctional cytokine production and degranulation, resulting in less effective cytotoxicity toward tumor cells. Interestingly, a comparable dysfunctional phenotype is also inducible in Vγ9Vδ2 T cells yielded from healthy individuals when co-cultured with CLL cells, suggesting a leukemia-induced immune-dysfunction [42] . We have previously reported that the in vitro proliferative response of Vγ9Vδ2 T cells is significantly impaired in a subset of patients affected by CLL (low-responder, LR, patients), who also display an unbalanced Vγ9Vδ2 T-cell subset distribution in favor of effector memory and terminally differentiated effector memory cells [43] . The LR condition was associated with unmutated IGHV status and, most importantly, it was an independent predictor of shorter TTFT in multivariate analysis. These findings support the concept that tumor-induced chronic activation fosters the undesired accumulation of immune cells inadequate for an effective antitumor activity.

Tregs are a fundamental CD4+ T-lymphocyte subset which prevents excessive immune activation and thus autoimmunity. In regards to hematological malignancies, Tregs may contribute to a pro-tumoral microenvironment, facilitating tumor progression [44] . Several studies demonstrated an increased count of circulating Tregs in CLL patients compared to healthy controls [45] [46] [47] [48] . Interestingly, the expansion of Tregs is probably mediated by CD27-CD70 interactions and by a resistance to apoptosis, rather than by chronic antigenic stimulation [49] . Higher Treg numbers have been associated with increased tumor load and advanced stages of disease [45, [50] [51] [52] . Specifically, Treg count progressively increases as the patients transition from an healthy status, to a monoclonal B cell lymphocytosis (MBL), and later to an overt and advanced CLL [53] ; furthermore, Treg count is higher in patients with progressive disease compared to those with a stable disease [14, 28, 46, 54] . Treg count has also been associated with CLL-related prognostic markers such as CD38 and ZAP70 expression [55] , and in CLL, higher Treg absolute number and frequency correlate with shorter TTFT and OS [47, 56, 57] . Notably, different non-chemotherapy drugs such as thalidomide, lenalidomide, idelalisib and ibrutinib have shown the ability to restore or at least reduce Treg counts [58] [59] [60] .

Beside the increased count, Tregs from CLL patients produce a larger amount of IL-10 and TGF-β1 and overexpress CTLA-4 compared to healthy controls [33, 52, 61] . Motta et al. showed in a cohort of 40 untreated patients with CLL, that CTLA-4 expression in CD4+CD25+ T cells is increased and correlates with advanced Rai stage, hypogammaglobulinemia, adverse cytogenetics and unmutated IGHV status [33] .

Finally, the prognostic impact of Tregs in CLL has also been assessed in relation to other T-cell subsets. Specifically, a reduced Tregs/Th17 ratio due to Th17 number increase has been associated with autoimmune cytopenias [24] . Also, a higher Treg number has been correlated to dysfunctional Vγ9Vδ2 T lymphocytes in untreated CLL patients [43] . Taken together, all these observations suggest an active role of Tregs in the progression of the CLL.

Natural Killer (NK) cells are fundamental components of the innate immune system and play an important role in antitumor immunity, being able to kill neoplastic cells without priming or prior activation [1, 62] . However, the activity of NK cells, which is regulated by activating and inhibitory signals, is disrupted in malignant environments [63] . Specifically, in CLL, leukemic cells can interact directly with the host's lymphocytes and secrete cytokines that alter the number, subset distribution and functions of NK cells [64] .

Quantitative NK abnormalities have shown to impact both prognosis and therapeutic efficacy in CLL. Different groups have confirmed that, when compared to healthy donors, patients with CLL univocally present higher NK-cell count [8, 9, 65, 66] . Interestingly, increased NK-cell count (>0.40 × 10 9 /L) corresponds to positive prognostic factors (i.e., early Rai or Binet stage, normal levels of β2-microglobulin, negativity of ZAP70 or CD38 expression, absence of TP53 gene mutation, mutated IGHV status and absence of ATM deletion) [66] . Also, the NK-cell count does not seem to decrease with disease progression [65] . However, no correlation between NK-cell absolute number and TTFT was reported [8, 9, 66] and, in regard to OS prediction, only Wang et al. have correlated a shorter OS to a lower NK-cell count [66] . When examining NK-cell relative number, a higher NK:MBC ratio was associated to low-risk disease (i.e., early Rai stages and mutated IGHV status) [8, 9] , but only Palmer et al. correlated NK-cell relative number to a longer TTFT [8] .

The lack of a strong prognostic correlation, even though NK-cell number is consistently increased in patients with CLL, may be attributable to the NK-cell dysfunctional cytotoxicity [65] . In line with this hypothesis, NK cells from patients with CLL have shown an impaired expression of the NKG2D costimulatory receptor and, consequently, a defective cytotoxic activity [65] . Along with quantitative defects, MBC can in fact also cause functional aberrations in the NK-cell compartment (briefly summarized in Table 1) , thus affecting NK cell-mediated immune mechanisms that are innately able to recognize cancer cells and which are also often exploited by therapeutical approaches. Amongst the latter, we find anti-CD20 monoclonal antibody (mAb)-based therapies, whose anti-tumor activity is strongly linked to the NK cell-related antibody-dependent cell-mediated-cytotoxicity (ADCC). Multiple pieces of evidence support the hypothesis that NK-cell number and activity have a direct impact on the efficacy of specific treatment regimens. As an example, Vitale et al. showed that, in patients with CLL treated with a regimen containing the anti-CD20 mAb ofatumumab and lenalidomide, those who achieved a complete response had a higher baseline absolute number and a more preserved function of NK cells, as compared to those who did not respond [67] . Furthermore, in CLL the effectiveness of anti-CD20 mAb treatments is positively affected by recombinant human IL-15 and lenalidomide, which are able to induce NK-cell proliferation and to improve NK cells-mediated ADCC and cytotoxicity [65, 66, 68] . Table 1 . Relevant qualitative NK-cell aberrations reported in patients with CLL.

Consequence Impact on Disease Ref.

Reduction of NKp30 and NKp46 activating receptors Exhaustion state on NK cells Immune escape [63] Increased expression of Tim-3 immune checkpoint Exhaustion state on NK cells Immune escape [63] Abundance of immature CD56 bright NK cells Reduced NKG2D activating receptor and cytokine (IL-10 and IL-13) secretion CLL cells survival and proliferation [69, 70] Irregular NKG2AR activity and reduced killer Ig-like receptors (KIRs)

Hampering of NK-cell cytotoxicity and viability Compromised immune system [1] Lower natural cytokine receptors (NCRs) expression Immune escape [69] NKG2D downregulation Hampering of NK cytotoxicity [65] 2.

Another important player of the immune surveillance against tumors are type I invariant natural killer T (NKT) cells, which recognize glycolipid antigens presented by CD1d, an MHC class I-like molecule, expressed also on CLL cells [71, 72] . A lower frequency of circulating NKT cells were found in CLL patients compared to healthy controls [72] and were correlated with progressive disease [73] . Indeed, the reduction of NKT-cell number negatively affects antitumor response, as it causes lower TNF expression and a decrease in the production of IFN-γ and cytokines involved in T-and NK-cell activation [74] . In CLL patients, CD1d is expressed on malignant B cells in a lower percentage comparted to normal B cells [73] . However, CD1d expression increases along with disease progression and correlates with unfavorable prognostic factors [i.e., higher ZAP70 and CD38 expression, presence of del(11q) and/or del(17p)] and shorter TTFT and OS [72] . Similarly, Gorini et al. showed in 46 patients with CLL a significant correlation between lower NKT-cell number and higher numbers of malignant CD1d high cells with disease progression [75] . Along with numerical abnormalities, a functional impairment of NKT cells isolated from CLL patients with high CD1d expression was also reported, possibly due to progressive exhaustion of NKT cells following chronic stimulation from CD1d high CLL cells [75] . To date, little is known on how NKT cells can affect therapeutical approaches in CLL, however it has been reported that, even if decreased in number, they are not functionally impaired after chemotherapy [76] . Therefore, therapies aiming at enhancing NKT cells in CLL may be a possible advantageous tool to reinforce antitumor immunity in CLL.

Regarding normal B-cell dysfunctions, most available data focus on immunoglobulin (Ig) deficiency, which results from CLL cells inhibition on the residual subset of normal B cells. Hypogammaglobulinemia is a common condition in patients with CLL, with a frequency ranging from 20% to 70% of cases, depending on the heterogeneity of the analyzed populations [77] [78] [79] [80] . Data regarding the impact of hypogammaglobulinemia on infection rates are controversial. Although several groups established an association between the presence of hypogammaglobulinemia and the occurrence of infections [81] [82] [83] , few reports showed that Ig deficiency does not specifically correlate with the incidence of infections, but rather with an increased risk of deaths from all causes [84, 85] , thus indicating hypogammaglobulinemia as a marker of leukemia-induced microenvironment alteration and, in general, of disease aggressiveness. In line with this concept, the presence of Ig deficiency was reported to correlate with more advanced stages of disease (i.e., Rai III-IV and Binet B-C) [86] [87] [88] and with patients' high-risk features (i.e., unmutated IGHV or unfavorable cytogenetics) [88, 89] .

The overall impact of hypogammaglobulinemia on the OS of patients with CLL remains unclear. In a cohort of 159 newly diagnosed patients, Andersen and colleagues reported that any type of Ig deficiency is an adverse prognostic factor for OS [85] . Conversely, data from the Israeli CLL study group, including 1113 Binet stage A patients, and from the Mayo Clinic group, including 1482 newly diagnosed patients, described no significant association between hypogammaglobulinemia and OS [88, 90] . In terms of Ig classes, IgA deficiency seems to be a strong negative predictor, as shown by the association of reduced IgA levels with shorter TTFT, treatment-free-survival (TFS) and-in some cases-OS [79, [89] [90] [91] [92] [93] . Interestingly, the ability of ibrutinib administration to improve serum IgA levels -with an observed lower rate of infections in patients showing greater improvements in IgA -further confirmed the role of IgA as both an active weapon against infections and an indicator of improved immune functionality [94] . Concerning the prognostic impact of other Ig classes, decreased IgM levels have been reported to predict shorter TFS and OS [85] , while IgG deficiency, and especially low levels of IgG1 and IgG3 subclasses, have been associated with shorter TFS and OS in univariate analysis [87] . The main reason for the controversy of these observations lies in the retrospective nature of most studies, which enrolled heterogeneous and often not comparable patient populations, mainly consisting of early-stage patients. Further prospective studies with a more balanced distribution of patients in terms of disease stage and indications to treatment will be certainly instrumental in the definition of the impact of Ig deficiencies on patients' outcome.

In conditions of chronic inflammation, monocytes, macrophages and dendritic cells (DCs) differentiate within the inflammatory environment and acquire immunosuppressive characteristics, thus becoming myeloid-derived suppressor cells (MDSCs). This reprogram-ming process is induced by several factors, some of which derive directly from tumor cells and allow immune escape [95] . In the setting of CLL, the crosstalk between cancer cells and their myeloid microenvironment has been extensively studied. Indeed, patients with CLL present an increased number of CD14+HLA-DR low monocytes [96] [97] [98] . This correlates-from the biologic standpoint-with decreased antigen-presenting capacity and decreased immune-stimulatory capacity of monocytes [96] and -from the clinical standpoint -with advanced Rai stages (i.e., III and IV) [99] . Moreover, higher monocytic MDSCs (M-MDSCs) count was noticed in patients presenting high-risk disease features [i.e., ZAP70 positivity, presence of del(11q) and/or del(17p), or unmutated IGHV] [99] and correlated with shorter time to progression (TTP), TTFT and OS [97, 99, 100] .

Another key player of myeloid origin in the tumor microenvironment are the nurse like cells (NLCs), which are tumor-associated macrophages exhibiting M2 hallmarks and secreting immunosuppressive and tumor-supportive cytokines [101] . NLCs induce apoptosisresistance in tumor cells [102] , and elevated serum levels of NLCs-secreted cytokines in patients with CLL correlate with poor prognostic factors (i.e., Rai stage III or IV, elevated LDH and/or β2-microglobulin) and have a negative impact on OS [103, 104] .

Although little is known on how these alterations of the tumor microenvironment can affect treatment results, a recent study has reported on the impact of ibrutinib on the myeloid compartment. Ferrer et al. showed that after treatment initiation, polymorphonuclear MDSCs (PMN-MDSCs) numbers progressively decline, whereas M-MDSC numbers are unaffected. Notably, this ibrutinib-induced reduction in PMN-MDSCs on one hand drove the differentiation of T lymphocytes toward less immunosuppressive Th cells, and on the other hand correlated with a decrease in CLL cell number and with a clinical improvement [100] . Based on these observations, immunotherapeutic strategy lowering the number of MDSCs, and more specifically of PMN-MDSCs, might be a valuable option for treatment of patients with CLL.

Beside hypogammaglobulinemia, other alterations of the humoral immunity have been broadly described in CLL patients. For instance, exhaustion of the classical pathway of the complement cascade has been reported in 38% of patients, resulting in a hampered complement-dependent cytotoxicity that may affect both protection from infections and the therapeutic efficacy of mAbs [105] . Interestingly, an impact of changes in the complement cascade on CLL prognosis has already been suggested. Indeed, Varga et al. have shown that a low activity of the classical complement pathway at CLL diagnosis predicts a short OS, especially in patients with Rai stage II and III disease [106] .

Another peculiar aspect of the altered humoral immunity in CLL is the presence of a specific cytokine signature, capable of influencing the course of the disease [107] . In CLL, cytokines are mainly produced by leukemic cells, but they may also originate from the interactions between the neoplastic clone and immune cells (i.e., T cells, NLCs, bone marrow stromal cells) [108] . The modulation of the cytokine milieu within the tumor can in turn stimulate the growth and survival of the neoplastic clone [109] , through the receptormediated activation of different signaling pathways involved in cell migration, proliferation and apoptotic function. Over the years, the altered expression of several cytokines has been correlated with CLL biological characteristics and prognostic features ( Table 2) . Cytokine profiling of patients' sera has shown that increased levels of Th2-related cytokines and decreased levels of Th1-related cytokines correlate with an aggressive disease and predict the need of therapy [110] . Notably, in line with previous data disclosing a correlation between serum concentration of some chemokines and CLL prognosis [111] , Yan and colleagues identified a panel of 17 cytokines/chemokines that were significantly elevated in CLL patients compared to healthy controls [112] . Among these, a group of chemokines specifically involved in cell migration and in T-cell attraction and infiltration have demonstrated to predict a worse prognosis. More recently, in a study by Agarwal and colleagues, six cytokines (i.e., SDF-1/CXCL12, uPAR, IGFBP-2, BMP-4, MCP-4, IL-1 R4/ST2) resulted to be statistically different between initial and advanced stages of the disease [113] . Of those, SDF-1, a chemoattractant for leukemic B cells expressing CXCR4, resulted to be overexpressed in patients with advanced disease [113] . Our group has recently demonstrated that the SDF-1/CXCR4 axis is critically involved in the microenvironment-induced protection of CLL cells through the positive regulation of HIF-1α transcription factor [114] . Interestingly, we also reported that HIF-1α, which regulates tumor cell adaptation to hypoxia and to microenvironmental stimuli, is overexpressed in CLL cells from patients carrying IGHV unmutated genes and TP53 alterations, who are typically characterized by poor outcome and resistance to therapy [115, 116] . Of note, the overexpression of chemokine receptors, such as CXCR4, on CLL cells has prompted the investigation of new potential therapeutic targets. Unfortunately, despite showing significant activity against CLL cells, none of CXCR4-inhibitors has so far been transferred to clinical practice [117] [118] [119] . * IL-6 and IL-10 emerged as independent prognostic factors for OS both when analyzed individually and in combination. § The cytokine low-risk group comprised patients with either low TNFα or low IL-10 or those with only one elevated parameter. The cytokine high-risk group comprised patients with both high TNFα and high IL-10.

The complex immune alterations characterizing CLL eventually manifest in clinically relevant immune dysfunctions, including autoimmune phenomena and increased risk of infections [138] [139] [140] [141] . Also, this immune dysregulation leads to an increased risk of second malignancies in patients with CLL [142, 143] . These additional clinical manifestations are overall the most common complications that affect the course and management of this chronic disease, and they impact on the overall CLL prognosis.

Autoimmune phenomena frequently complicate the clinical course of patients affected by CLL. As reviewed in [141] , up to a quarter of patients with CLL may present concomitant autoimmune manifestations, which are primarily autoimmune cytopenias, including autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), pure red cell aplasia and autoimmune granulocytopenia. Conversely, non-hematological autoimmunity, such as Hashimoto's thyroiditis, rheumatoid arthritis, vasculitis, bullous pemphigus or acquired angioedema, are undoubtedly rarer.

Multiple immune mechanisms are involved in the pathophysiology of CLL-related autoimmune manifestations, and both humoral and cellular immune dysfunctions may support the development of these complications [141] . Interestingly, autoimmune cytopenias appear to occur most frequently in patients with CLL in advanced clinical stage, whereas non-hematological autoimmune complications are more common in the initial phases of the disease, possibly suggesting an even wider heterogeneity in the pathogenic mechanisms [144, 145] .

The observed association of autoimmune cytopenias with the disease stage may also be determined by the fact that the Binet and Rai staging systems do not discriminate between bone marrow infiltration and autoimmunity as the cause of anemia or thrombocytopenia [146] . Different groups have shown that the survival for patients with anemia or thrombocytopenia of autoimmune origin is longer than for those with cytopenias attributable to bone marrow infiltration [147] [148] [149] . Beside the clinical stage, a significant association between CLL-related autoimmune complications and other negative prognostic parameters, such as high lymphocyte count, high β2-microglobulin or LDH level, increased CD38 or ZAP-70 expression, adverse FISH [i.e., del(17p) or del(11q)], or unmutated IGHV has been observed in different cohorts [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] . Of note, not all prognostic parameters were assessed by all groups, and-most importantly-not all publications are concordant in reporting an association between autoimmune manifestations and some of the variables, possibly depending on the heterogeneity of the cohorts evaluated, on the retrospective nature of the studies and on the variability in the criteria used to define autoimmune cytopenias.

In spite of the frequently reported association of autoimmune cytopenias with adverse prognostic factors, the number of articles specifically concluding for a significant impact of autoimmune phenomena on the prognosis of patients with CLL is limited. In their cohort of 473 consecutively diagnosed patients with CLL, Visco and colleagues reported a 7% occurrence of AIHA: there was not a significant difference in terms of OS between patients with or without AIHA, but patients developing AIHA earlier in the disease course (i.e., within 48 months after CLL diagnosis) had a significantly inferior OS compared to those with late-onset AIHA or to those who did not develop AIHA at all [159] . Sevenhundred seventy-seven patients with treatment-naive CLL requiring therapy, who were enrolled in the randomized UK LRF CLL4 trial, were evaluated by Dearden et al. [160] . Among patients who were tested for direct antiglobulin test (DAT) at study entry, 89 (14%) resulted positive, and among those with available information, 77 (10%) developed AIHA during treatment. Both DAT positivity and the development of AIHA were predictive for shorter PFS and OS in this cohort. Accordingly, other groups confirmed the negative prognostic impact of DAT positivity at CLL diagnosis, independently from the occurrence of AIHA [161] [162] [163] .

In a large cohort of 1477 patients with CLL, Shvidel et al. identified 100 patients with autoimmune cytopenia (7%) [164] . As compared with control patients without cytopenia and who never developed AIHA or ITP, patients with AIHA had a significantly worse outcome in terms of OS from the time of CLL diagnosis, whereas the survival was similar for patients with ITP and controls. Conversely, in a cohort of 1278 newly diagnosed patients with CLL, 64 cases of ITP were identified (5%) and the development of the autoimmune phenomenon at any time during the disease course conferred a shorter OS [152] . In these patients, an early occurrence of ITP (i.e., within 24 months after CLL diagnosis) was found to be an independent adverse prognostic factor for OS. As for the association with prognostic parameters, the evaluation of the impact of autoimmunity on OS also produced inconsistent results, mainly due to the heterogeneity of the patient populations analyzed in different studies. Notably, the prognostic impact of non-hematological autoimmune manifestations is even more difficult to ascertain due to the rarity of their occurrence.

It is widely accepted that the initial treatment for CLL-associated autoimmune cytopenias is based on steroids, possibly in combination with rituximab and/or immunosuppressive drugs, whereas when CLL treatment criteria are fulfilled or when the autoimmune phenomenon is not controlled, a CLL-directed treatment is recommended [146, 165] . Few data are available regarding the impact of autoimmune manifestations on response to CLLdirected therapy. In the previously mentioned UK LRF CLL4 trial reported by Dearden and colleagues, where patients were randomized to receive chlorambucil, fludarabine or fludarabine plus cyclophosphamide, it was noted that patients who developed AIHA had inferior response rates and quality [160] . Of note, patients with AIHA did receive less therapy. More recently, the presence of a positive DAT was found to be a predictor for non-response to frontline chemoimmunotherapy treatment in a cohort of 120 patients with CLL [166] .

The therapeutic scenario for CLL recently changed, with the introduction of targeted agents, such as B-cell receptor inhibitors and Bcl-2 antagonists [167] . Interestingly, data from our group and others indicate that in patients treated with ibrutinib, idelalisib or venetoclax, a pre-existing autoimmune cytopenia does not carry an adverse impact on patients' prognosis, suggesting that these effective drugs might be able to attenuate the inferior outcome associated with autoimmune manifestations [156, 158] .

In patients with CLL, infection is a common cause of morbidity and mortality, accounting for up to more than 50% of the deaths-depending on the characteristics of the patients' cohorts included in the different studies-and the negative impact of infections raises in patients with multiple comorbidities [85, 168, 169] . Interestingly, as compared to a control population, the risk of infection is higher not only in patients with CLL, but also in those with MBL, the pre-disease stage preceding CLL [170] . The increased risk of infection is certainly dependent on the underlying CLL-related immune dysfunctions, but also on the immune perturbations specifically related to different CLL-directed therapies. Accordingly, current guidelines recommend antimicrobial prophylaxis in patients with a higher risk of developing infections, based on the treatment regimen received [165] .

The possible correlation of infections and CLL prognostic parameters has been explored. Francis at al. retrospectively evaluated a cohort of 280 patients, and-as expectedinfection rate inversely associated with Ig levels [81] . Also, patients with advanced clinical stage, unmutated IGHV, genetic abnormalities [TP53 mutation or deletion, ATM mutation or deletion, tris (12) ] and those with CD38 positivity had a shorter time-to-first major infection, with both stage and IGHV mutational status maintaining their independent value in multivariate analysis. The same four parameters negatively impacted infection-related mortality, possibly reflecting an association between disease aggressiveness and immune deficiency. Consistently, in the monocentric cohort of 706 patients reported by Visentin and colleagues, major infections-defined as events requiring in patient management or intravenous antibiotics-were associated with clinical stage, IGHV mutational status, high-risk cytogenetics and CD38 positivity [80] . More recently, Andersen and colleagues interrogated the Danish National CLL registry, assessing a cohort of 2905 patients diagnosed with CLL between the years 2008 and 2016 [82] . In multivariate analysis, variables that significantly associated with infection-specific hazard rates were Binet stage, β2-microglobulin and IgA levels. As highlighted by the authors, the lack of association of the risk of infections with adverse FISH abnormalities or unmutated IGHV status might be explained by the impact of these variables on the probability of treatment, which was a competing risk in this model.

Undoubtedly, the development of models capable to identify patients more susceptible of developing severe infections during the course of their disease would be extremely useful, also in terms of treatment selection. To this aim, Agius and colleagues recently developed a machine learning model identifying with high precision patients at risk of severe infection within 2 years of CLL diagnosis [171] .

Regarding the evaluation of the impact of infections on the overall prognosis of patients with CLL, in the already cited Danish registry cohort, patients with CLL who have had an infection during the first year after diagnosis-identified as those who had a blood culture drawn prior to CLL therapy-had a significantly shorter TFS and OS [172] . In their monocentric cohort of 706 patients, Visentin and colleagues showed that patients with a history of major infection had a shorter OS compared to those who did not experience this complication, and this factor maintained its impact on survival in multivariate analysis [80] . Data from Crassini et al., who analyzed the long-term follow-up (9.5 years) of a cohort of 147 patients, confirmed a significant association between the occurrence of serious or recurrent infectious complications within the first year of observation and a shorter OS [173] .

It needs to be highlighted that in the last few years, the introduction of targeted agents for the treatment of patients with CLL may have had an impact on the infectious complication spectrum for these patients (reviewed in [140] ). For example, opportunistic pathogens have been recognized as an emerging cause of infection in patients treated with ibrutinib. However, the infectious risk seems more related to the disease itself and to the previous treatment status than to the administered drugs. Recently, Mauro and colleagues specifically evaluated the prognostic impact of infections in 494 patients with CLL treated with ibrutinib [174] . Ibrutinib was permanently discontinued in 9% of patients due to infections and OS for patients who had a severe infection or pneumonia was significantly shorter compared to those infection-free.

Finally, it is worth to be mentioned that in the current worldwide pandemic scenario, patients with CLL can be severely affected by SARS-CoV-2 infection [175] . Older patients seem to be at increased risk of infection, with a high incidence of mortality among hospitalized patients (reviewed in [176] ). Interestingly, it has been postulated that BTK inhibitors such as ibrutinib or acalabrutinib could attenuate hyperinflammatory responses thus exerting protection against severe disease course, but to date data are still controversial and prospective clinical trials are ongoing [176] ).

Considering the high median age at diagnosis (i.e., 70 years) of patients with CLL [177] , who mostly remain under surveillance for years, it is not surprising how during the course of the disease the onset of another cancer often occurs, and has a considerable impact on the patient's prognosis. Strati et al. reported their results on a prospective cohort of 1143 newly diagnosed patients with CLL, amongst whom 225 deaths were reported after a median follow-up of 6 years: cause of death was attributed to a second malignancy in 19% of patients [168] . Undoubtedly, together with the age, CLL-related factors-such as the loss of immune surveillance and previous treatments-exert their contribute on the occurrence of other malignancies. Registry-derived data from the early 1990s already reported a significantly increased risk of developing a second malignancy in patients with CLL, compared to the general population [178] .

The contribution of previous treatments in this increased risk has been specifically evaluated by Falchi and colleagues, who analyzed the impact of other malignancies in 797 patients with CLL who survived >10 years [143] . In this cohort, an excess of cancer diagnosis in patients with CLL compared to the general population was confirmed, with a standardized incidence ratio (SIR) of 1.2. Interestingly, the cumulative frequency of other cancers was similar in patients who received treatment for CLL and in those who remained untreated (36%), and the therapy for CLL was not associated with the occurrence of other malignancies in multivariate analysis. Conversely, a large population-based analysis on data from 38754 patients with CLL, derived from the Surveillance Epidemiology and End Results (SEER) database, showed an increased risk of second primary malignancies in patients who had received prior chemotherapy compared to those untreated or with an unknown treatment status (SIR 1.38 vs. 1.16) [179] . However, when the analysis was restricted to patients diagnosed with CLL in the time interval 2003-2015-when an overall increasing trend of second primary malignancies was noted-no difference was found in the risk for solid tumors between previously treated and untreated patients.

Besides the impact of CLL-directed treatments, some attempts have been made to identify a possible correlation between the development of second cancers and CLLrelated biological parameters. In their retrospective analysis on 2028 patients with CLL, Tsimberidou and colleagues reported that elevated levels of β2-microglobulin and LDH, but not the presence of cytogenetic aberrations, were independent factors predicting for the development of second cancers [180] . However, in the already cited analysis on long-term survivors performed by Falchi et al., β2-microglobulin did not emerge as independent predictor, possibly reflecting differences in the patients' cohorts [143] . Additional studies should assess the impact of risk factors, including immune parameters, on the risk of developing second primary malignancies.

The group at Mayo Clinic specifically focused on the risk of developing a second lymphoproliferative disorder, retrospectively analyzing 962 patients with CLL [181] . After a median follow-up of 3.3 years, 2.9% of patients developed a second lymphoproliferative disorder, and this was not associated with CLL biological characteristics, such as ZAP70 or CD38 expression, IGHV mutational status or cytogenetic aberrations. The incidence of and risk factors for second malignancies was also recently evaluated in a cohort of 691 patients treated with ibrutinib or acalabrutinib [182] . After a median follow-up of 44 months, 20% of patients were diagnosed with a non-melanoma skin cancer and 9% with other primary malignancies, which were responsible for 13% of deaths. In this cohort, the SIR for secondary invasive cancers was 2.2, and a lower risk for other cancers was significantly associated in multivariate analysis with a higher baseline CD8+ T-cell count, supporting a correlation between immune function and carcinogenesis in this patient population.

It is conceivable that the presence of another malignancy may impact the overall prognosis of a patient. In the previously cited analysis from Tsimberidou et al., patients who had a history of a prior malignant neoplasms at the time of presentation with CLL had a significantly shorter OS as compared to those who did not. However, the authors did not specifically evaluate the survival of patients who developed a second malignancy after CLL diagnosis compared to those who did not [180] . Among 12,041 patients with CLL from the Swedish Cancer Registry, Toro and colleagues reported 236 cases of non-melanoma skin cancer, including 111 squamous cell cancers [183] . These patients had a significantly shorter OS than CLL patients without non-melanoma skin cancer, and 44% of deaths were attributed to CLL. However, it has to be highlighted that the median age at CLL diagnosis for patients with a non-melanoma skin cancer was significantly higher compared to those without a history of non-melanoma skin cancer (78.5 vs. 71 years). Finally, Royle et al. reported in their cohort of 13580 patients diagnosed with CLL in Australia between 1983 and 2005 a SIR for second primary cancers of 2.17 [184] . Sixty-five% of deaths were attributed to cancer (15% excluding lymphoproliferative neoplasms). Overall, CLL patients had a 2.5 times higher mortality rate as compared to the general population, and their age standardized cancer mortality ratios-excluding lymphoproliferative neoplasms-was 1.72. Additional studies are currently needed to elucidate whether second primary malignancies have a more aggressive evolution, ultimately leading to a worse outcome, in patients with CLL compared to the general population.

The evaluation of the multiple immune alterations occurring in patients with CLL, which affect both the innate and adaptive immunity, is certainly fundamental to better understand the biology of the disease. Some of these immune system dysfunctions are associated with other disease-specific prognostic hallmarks and have been shown to have a prognostic impact. These observations support the notion that the clinical heterogeneity that characterizes CLL depends not only on the intrinsic features of the tumor clone, but also on the complex interrelation occurring between the cancer cells, the immune system and the tumor microenvironment.

The disease-related immune dysfunctions can be further exacerbated by the immunosuppressive effects of the CLL-directed therapies, constituting a double-hit process that favors the development of clinically relevant manifestations such as autoimmunity, infections and second malignancies. These manifestations may have an impact on the overall patients' prognosis, not only because they directly impact the clinical outcome, but also because they easily interfere with the treatment program, causing delays or interruptions. Of note, in some patients, autoimmune phenomena, infections or second cancers can even occur simultaneously, on one hand because of a higher susceptibility linked to the intrinsic features of higher-risk subtypes of the disease, and on the other hand because of the mutual influence that these three complications can exert on each other: in patients with autoimmune cytopenia, infections are a frequent complication following immunosuppressive treatments, and second malignancies have been reported as the leading cause of death [147] . The use of less toxic treatments, exerting a reduced impact on the immune system functionality, may be beneficial on the overall management of these patients, especially when other comorbidities are present.

Most of the data presented in this article were collected from the chemotherapy or chemoimmunotherapy era, and the advent of targeted agents may arguably result in changes in the current scenario. Therapies with an improved efficacy prolong patients' survival and confer a longer follow-up duration, thus increasing the possibility to detect disease-related complications over a longer period of time. Targeted drugs-which along with their anti-tumor activity exert multiple off-target effects on different components of the immune system and tumor microenvironment (reviewed in [1, 2, 185, 186] )-may also directly impact on the risk of immune dysfunctions and immune-dependent clinical manifestations. Therefore, we expect that the current wide use of these compounds in the clinical practice and the progressively growing follow-up duration will provide more data and pieces of information to be added to this complex scenario. 

Immune Dysfunctions and Immune-Based Therapeutic Interventions in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukaemia: The role of T cells in a B cell disease

T-cells in chronic lymphocytic leukemia: Guardians or drivers of disease?

Unusual T-Cell Phenotype in Advanced B-Chronic Lymphocytic Leukaemia

Abnormal T lymphocyte subpopulations in patients with B cell chronic lymphocytic leukemia: An analysis by monoclonal antibodies

Imbalance of T cell subpopulations in patients with chronic lymphocytic leukaemia of the B cell type

Disease-Specific Complications of Chronic Lymphocytic Leukemia

Prognostic importance of T and NK-cells in a consecutive series of newly diagnosed patients with chronic lymphocytic leukaemia

Prognostic significance of CD8 and CD4 T cells in chronic lymphocytic leukemia

Control of chronic lymphocytic leukemia development by clonally-expanded CD8 + T-cells that undergo functional exhaustion in secondary lymphoid tissues

CLINICAL SIGNIFICANCE OF T-CELLS IN CHRONIC LYMPHOCYTIC LEUKAEMIA

Prognostic significance of immune function parameters in patients with chronic lymphocytic leukaemia

Phenotypic alteration of CD8+ T cells in chronic lymphocytic leukemia is associated with epigenetic reprogramming

Expansion of a CD8 + PD-1 + replicative senescence phenotype in early stage CLL patients is associated with inverted CD4:CD8 ratios and disease progression

Increased frequency of CD4+PD-1+HLA-DR+ T cells is associated with disease progression in CLL

Prognostic role of CD4 T-cell depletion after frontline fludarabine, cyclophosphamide and rituximab in chronic lymphocytic leukaemia

T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers

The PD-1/PD-L1 axis contributes to T-cell dysfunction in chronic lymphocytic leukemia

T type 1/type 2 subsets balance in B-cell chronic lymphocytic leukemia-The three-color flow cytometry analysis

TBET-expressing Th1 CD4+ T cells accumulate in chronic lymphocytic leukaemia without affecting disease progression in Eµ-TCL1 mice

Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD81 T-cell responses

TH2/TH1 Shift Under Ibrutinib Treatment in Chronic Lymphocytic Leukemia

IL-17 Signaling in the Tumor Microenvironment

Regulatory T-cell and T-helper 17 balance in chronic lymphocytic leukemia progression and autoimmune cytopenias

Th17 and non-th17 interleukin-17-expressing cells in chronic lymphocyticleukemia: Delineation, distribution, and clinical relevance

Th17/IL-17A might play a protective role in chronic lymphocytic leukemia immunity

Identification and characterization of distinct IL-17F expression patterns and signaling pathways in chronic lymphocytic leukemia and normal B lymphocytes

Downregulation of IL-17-producing T cells is associated with regulatory T cell expansion and disease progression in chronic lymphocytic leukemia

Clonal Expansion Within the CD4+CD57+ and CD8+CD57+ T Cell Subsets in Chronic Lymphocytic Leukemia

Peripheral blood lymphocyte subset shifts in patients with untreated hematological tumors: Evidence for systemic activation of the T cell compartment

Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells

High-dimensional mass cytometry analysis revealed microenvironment complexity in chronic lymphocytic leukemia

Increased expression of CD152 (CTLA-4) by normal T lymphocytes in untreated patients with B-cell chronic lymphocytic leukemia

Lymphocyte activation gene 3: A novel therapeutic target in chronic lymphocytic leukemia

Asgarian-Omran, H. Frequency and functional characterization of exhausted CD8 + T cells in chronic lymphocytic leukemia

TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

T cells from CLLpatients exhibit features of T-cell exhaustion but retain capacity for cytokine production

Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia

Non-MHC-restricted cytotoxic cells: Their roles in the control and treatment of leukaemias

The Role of Human γδ T Cells in Anti-Tumor Immunity and Their Potential for Cancer Immunotherapy

Vγ9Vδ2 T cell-based immunotherapy in hematological malignancies: From bench to bedside

Improving CLL Vγ9Vδ2-T-cell fitness for cellular therapy by ex vivo activation and ibrutinib

Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells

Regulatory T cell targeting in cancer: Emerging strategies in immunotherapy

Regulatory T-cell number is increased in chronic lymphocytic leukemia patients and correlates with progressive disease

Increased frequency of CD8+ and CD4+ regulatory T cells in chronic lymphocytic leukemia: Association with disease progression

A study for proposal of use of regulatory T cells as a prognostic marker and establishing an optimal threshold level for their expression in chronic lymphocytic leukemia

Regulatory T Cells and Their Prognostic Relevance in Hematologic Malignancies

Enhanced formation and survival of CD4 + CD25 hi Foxp3 + T-cells in chronic lymphocytic leukemia

Quantitative and qualitative analysis of regulatory T cells in B cell chronic lymphocytic leukemia

Chronic lymphocytic leukaemia cells drive the global CD4 + T cell repertoire towards a regulatory phenotype and leads to the accumulation of CD4 + forkhead box P3 + T cells

PI3Kδ inhibition modulates regulatory and effector T-cell differentiation and function in chronic lymphocytic leukemia

Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T-cells in chronic lymphocytic leukemia

Immune Response Dysfunction in Chronic Lymphocytic Leukemia: Dissecting Molecular Mechanisms and Microenvironmental Conditions

Increased IL-10/IL-17 ratio is aggravated along with the prognosis of patients with chronic lymphocytic leukemia

Regulatory T cells predict the time to initial treatment in early stage chronic lymphocytic leukemia

A shorter time to the first treatment may be predicted by the absolute number of regulatory T-cells in patients with Rai stage 0 chronic lymphocytic leukemia

The high frequency of T regulatory cells in patients with B-cell chronic lymphocytic leukemia is diminished through treatment with thalidomide

Lenalidomide in the treatment of chronic lymphocytic leukemia

Ibrutinib restores immune cell numbers and function in first-line and relapsed/refractory chronic lymphocytic leukemia

Reduced frequencies and suppressive function of CD4 + CD25 hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine

Dysregulated Expression of Tim-3 and NKp30 Receptors on NK Cells of Patients with Chronic Lymphocytic Leukemia

Cell-mediated immunity in chronic lymphocytic leukemia

Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease

Elevated absolute NK cell counts in peripheral blood predict good prognosis in chronic lymphocytic leukemia

Ofatumumab and Lenalidomide for Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia: Correlation between Responses and Immune Characteristics

Natural killer cell subsets and natural killer-like T-cell populations in benign and neoplastic B-cell proliferations vary based on clinicopathologic features

Expression of natural killer cell activating receptors in patients with chronic lymphocytic leukaemia

NK cell dysfunction in chronic lymphocytic leukemia is associated with loss of the mature cells expressing inhibitory killer cell Ig-like receptors

Retinoic acid induction of CD1d expression primes chronic lymphocytic leukemia B cells for killing by CD8(+) invariant natural killer T cells

CD1d expression is higher in chronic lymphocytic leukemia patients with unfavorable prognosis

Reduced frequency of NKT-like cells in patients with progressive chronic lymphocytic leukemia

Natural killer-like T CD3+/CD16+CD56+ cells in chronic lymphocytic leukemia: Intracellular cytokine expression and relationship with clinical outcome

Invariant NKT cells contribute to chronic lymphocytic leukemia surveillance and prognosis

An emerging role for immune regulatory subsets in chronic lymphocytic leukaemia

Clinical staging and immunological findings in chronic lymphocytic leukemia

Serum Immunoglobulins and Lymphocyte Subsets in Chronic Lymphocytic Leukemia

Serum immunoglobulins in B-chronic lymphocytic leukemia.Natural history and prognostic significance

Clinical profile associated with infections in patients with chronic lymphocytic leukemia. Protective role of immunoglobulin replacement therapy

The effect of immunoglobulinVH gene mutation status and other prognostic factors on the incidence of major infections in patients with chronic lymphocytic leukemia

Incidence and predictors of infection among patients prior to treatment of chronic lymphocytic leukemia: A Danish nationwide cohort study

Early stage chronic lymphocytic leukaemia carrying unmutated IGHV genes is at risk of recurrent infections during watch and wait

Disease activity and pretreatment, rather than hypogammaglobulinaemia, are major risk factors for infectious complications in patients with chronic lymphocytic leukaemia

Hypogammaglobulinemia in newly diagnosed chronic lymphocytic leukemia is a predictor of early death

Clinical significance of serum immunoglobulin G subclass deficiency in patients with chronic lymphocytic leukemia. Scand

Humoral immune failure defined by immunoglobulin class and immunoglobulin G subclass deficiency is associated with shorter treatment-free and overall survival in Chronic Lymphocytic Leukaemia

Hypogammaglobulinemia in newly diagnosed chronic lymphocytic leukemia: Natural history, clinical correlates, and outcomes

IgA hypogammaglobulinemia predicts outcome in chronic lymphocytic leukemia

Serum immunoglobulin levels at diagnosis have no prognostic significance in stage A chronic lymphocytic leukemia: A study of 1113 cases from the Israeli CLL Study Group

Monoclonal gammopathy and serum immunoglobulin levels as prognostic factors in chronic lymphocytic leukaemia

Clinical relevance of hypogammaglobulinemia, clinical and biologic variables on the infection risk and outcome of patients with stage A chronic lymphocytic leukemia

IgA levels at diagnosis predict for infections, time to treatment, and survival in chronic lymphocytic leukemia

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib

The Role of Tumor-Associated Macrophages in Leukemia

Association of an increased frequency of CD14+ HLA-DR lo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)

Monocytic myeloidderived suppressor cells in chronic lymphocytic leukemia patients: A single center experience

CLL-cells induce IDOhi CD14+HLA-DRlo myeloid-derived suppressor cells that inhibit T-cell responses and promote TRegs

High M-MDSC Percentage as a Negative Prognostic Factor in Chronic Lymphocytic Leukaemia

Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL

Adenosine signaling mediates hypoxic responses in the chronic lymphocytic leukemia microenvironment

Sorafenib-induced apoptosis of chronic lymphocytic leukemia cells is associated with downregulation of RAF and myeloid cell leukemia sequence 1 (Mcl-1)

Beyond bystanders: Myeloid cells in chronic lymphocytic leukemia

Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia: Correlation with phenotypic characteristics and outcome

Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL

Low activity of the classical complement pathway predicts short survival of patients with chronic lymphocytic leukaemia

Clinico-Biological Implications of Modified Levels of Cytokines in Chronic Lymphocytic Leukemia: A Possible Therapeutic Role

Microenvironmental interactions and survival of CLL B-cells

A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis In Vitro

Aggressive disease defined by cytogenetics is associated with cytokine dysregulation in CLL/SLL patients

The role of chemokines in B cell chronic lymphocytic leukaemia: Pathophysiological aspects and clinical impact

Identification of outcome-correlated cytokine clusters in chronic lymphocytic leukemia

Genetic and cytokine changes associated with symptomatic stages of CLL

Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL

Simvastatin and downstream inhibitors circumvent constitutive and stromal cell-induced resistance to doxorubicin in IGHV unmutated CLL cells

HIF-1alpha is over-expressed in leukemic cells from TP53-disrupted patients and is a promising therapeutic target in chronic lymphocytic leukemia

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia

BMS-936564 / MDX1338): A fully human anti-CXCR4 antibody induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species-dependent pathway

Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells

Serum levels of CD8 antigen and soluble interleukin 2 receptors in patients with B cell chronic lymphocytic leukemia

Expression of soluble CD27 and interleukins-8 and -10 in B-cell chronic lymphocytic leukemia: Correlation with disease stage and prognosis

Increased autocrine interleukin-6 production is significantly associated with worse clinical outcome in patients with chronic lymphocytic leukemia

Association of interleukin-6 and interleukin-8 with poor prognosis in elderly patients with chronic lymphocytic leukemia

Clinico-biological implications of increased serum levels of interleukin-8 in B-cell chronic lymphocytic leukemia

Plasma interleukin 8 level predicts for survival in chronic lymphocytic leukaemia

Cytokine and adhesion molecule concentrations in blood of patients with B-cell chronic lymphocytic leukaemia with regard to disease progression

Role of high expression of IL-9 in prognosis of CLL

Evaluation of Interleukin-9 Expression as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia in a Cohort of Egyptian Patients

Interleukin-10 mRNA expression in B-cell chronic lymphocytic leukaemia inversely correlates with progression of disease

Serum tumor necrosis factor-α and interleukin-10 levels as markers to predict outcome of patients with chronic lymphocytic leukemia in different risk groups defined by the IGHV mutation status

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence

The clinical significance of tumor necrosis factor-alpha plasma level in patients having chronic lymphocytic leukemia

Expression of the chemokine receptors CXCR4 and CCR7 and disease progression in B-cell chronic lymphocytic leukemia/ small lymphocytic lymphoma

The prognostic role of CXCR3 expression by chronic lymphocytic leukemia B cells

The CX3C chemokine fractalkine (CX3CL1) is detectable in serum of B cell chronic lymphocytic leukemia patients with lymph node involvement

Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia

MIP-1α) plasma levels and the risk for disease progression in chronic lymphocytic leukemia

Perturbation of the normal immune system in patients with CLL

Infectious complications of chronic lymphocytic leukaemia: Pathogenesis, spectrum of infection, preventive approaches

Chronic lymphocytic leukemia and infection risk in the era of targeted therapies: Linking mechanisms with infections

Autoimmune Complications in Chronic Lymphocytic Leukemia in the Era of Targeted Drugs

Risk for second nonlymphoid neoplasms in chronic lymphocytic leukemia

Incidence and prognostic impact of other cancers in a population of long-term survivors of chronic lymphocytic leukemia

Relationship between autoimmune phenomena and disease stage and therapy in B-cell chronic lymphocytic leukemia

Clinical and serological autoimmune complications in chronic lymphocytic leukemia

iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL

Autoimmune cytopenia does not predict poor prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma

The prognostic significance of cytopenia in chronic lymphocytic leukaemia/small lymphocytic lymphoma

Autoimmune cytopenia in chronic lymphocytic leukemia: Prevalence, clinical associations, and prognostic significance

Autoimmune hemolytic anemia in chronic lymphocytic leukemia: Clinical, therapeutic, and prognostic features

Clinical and immunologic aspects of B chronic lymphocytic leukemia associated with autoimmune disorders

Impact of immune thrombocytopenia on the clinical course of chronic lymphocytic leukemia

Autoimmune cytopenia in chronic lymphocytic leukemia/small lymphocytic lymphoma: Changes in clinical presentation and prognosis

B-cell receptor configuration and adverse cytogenetics are associated with autoimmune hemolytic anemia in chronic lymphocytic leukemia

Major infections, secondary cancers and autoimmune diseases occur in different clinical subsets of chronic lymphocytic leukaemia patients

Autoimmune cytopenias in patients with chronic lymphocytic leukaemia treated with ibrutinib in routine clinical practice at an academic medical centre

The prevalence and prognostic significance of autoimmune cytopenias in a cohort of Egyptian patients with chronic lymphocytic leukemia

Pre-existing and treatment-emergent autoimmune cytopenias in patients with CLL treated with targeted drugs

Autoimmune hemolytic anemia in patients with chronic lymphocytic leukemia is associated with IgVH status

The prognostic significance of a positive direct antiglobulin test in chronic lymphocytic leukemia: A beneficial effect of the combination of fludarabine and cyclophosphamide on the incidence of hemolytic anemia

The negative prognostic significance of positive direct antiglobulin test in Chinese patients with chronic lymphocytic leukemia

Should a positive direct antiglobulin test be considered a prognostic predictor in chronic lymphocytic leukemia?

Old DAT and new data: Positive direct antiglobulin test identifies a subgroup with poor outcome among chronic lymphocytic leukemia stage A patients

Pathogenesis, prevalence, and prognostic significance of cytopenias in chronic lymphocytic leukemia (CLL): A retrospective comparative study of 213 patients from a national CLL database of 1518 cases

Chronic lymphocytic leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Revisiting Autoimmunity in Chronic Lymphocytic Leukemia: Prognostic Value of Positive Direct Antiglobulin Test in a Retrospective Study and Literature Review

Evolution of CLL treatment-From chemoimmunotherapy to targeted and individualized therapy

Relationship between co-morbidities at diagnosis, survival and ultimate cause of death in patients with chronic lymphocytic leukaemia (CLL): A prospective cohort study

Mapping comorbidity in chronic lymphocytic leukemia: Impact of individual comorbidities on treatment, mortality, and causes of death

Infectious complications among individuals with clinical monoclonal B-cell lymphocytosis (MBL): A cohort study of newly diagnosed cases compared to controls

Machine learning can identify newly diagnosed patients with CLL at high risk of infection

Immune failure, infection and survival in chronic lymphocytic leukemia in Denmark

Immune failure, infection and survival in chronic lymphocytic leukemia

Prognostic Impact and Risk Factors of Infections in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib

COVID-19 severity and mortality in patients with chronic lymphocytic leukemia: A joint study by ERIC, the European Research Initiative on CLL, and CLL Campus

Immune Dysfunction in Patients with Chronic Lymphocytic Leukemia and Challenges during COVID-19 Pandemic

Second cancers in patients with chronic lymphocytic leukemia

Trends in the risk of second primary malignancies among survivors of chronic lymphocytic leukemia

Other malignancies in chronic lymphocytic leukemia/small lymphocytic lymphoma

Risk factors for development of a second lymphoid malignancy in patients with chronic lymphocytic leukaemia

Second cancer incidence in CLL patients receiving BTK inhibitors

Prior history of non-melanoma skin cancer is associated with increased mortality in patients with chronic lymphocytic leukemia

Second cancer incidence and cancer mortality among chronic lymphocytic leukaemia patients: A population-based study

Harnessing the Effects of BTKi on T Cells for Effective Immunotherapy against CLL

Immunological changes with kinase inhibitor therapy for chronic lymphocytic leukemia