Moreno et al. J Exp Clin Cancer Res (2021) 40:321 https://doi.org/10.1186/s13046-021-02115-1 REVIEW Open Access Restoration of the immune function as a complementary strategy to treat Chronic Lymphocytic Leukemia effectively Carol Moreno1, Cecilia Muñoz2, María José Terol3, José‑Ángel Hernández‑Rivas4,5,6* and Miguel Villanueva7 Abstract Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy characterized by uncontrolled proliferation of B-cells and severe immune dysfunction. Chemo(immuno)therapies (CIT) have traditionally aimed to reduce tumor burden without fully understanding their effects on the immune system. As a consequence, CIT are usually associated with higher risk of infections, secondary neoplasms and autoimmune disorders. A better understanding of the biol‑ ogy of the disease has led to the development of therapeutic strategies which not only act against malignant B-cells but also reactivate and enhance the patient’s own anti-tumor immune response. Here, we review the current under‑ standing of the underlying interplay between the malignant cells and non-malignant immune cells that may promote tumor survival and proliferation. In addition, we review the available evidence on how different treatment options for CLL including CIT regimens, small molecular inhibitors (i.e, BTK inhibitors, PI3K inhibitors, BCL-2 inhibitors) and T-cell therapies, affect the immune system and their clinical consequences. Finally, we propose that a dual therapeutic approach, acting directly against malignant B-cells and restoring the immune function is clinically relevant and should be considered when developing future strategies to treat patients with CLL. Keywords: Immune function, Ibrutinib, CLL, Chronic Lymphocytic Leukemia Background In CLL, tumor cells influence the immune system to Chronic Lymphocytic Leukemia (CLL) is the most com- escape immunosurveillance and create an immunosup- mon B-cell malignancy in the Western world [1]. CLL is pressive microenvironment. In general, there are four a lymphoproliferative disease characterized by the accu- mechanisms used by CLL cells to escape from the con- mulation of mature monoclonal B-cells with a typical trol of immune cells: (1) non-immunogenic tumor cell immunophenotype (i.e, CD5+CD23+ and other B-cell death; (2) expansion and recruitment of immunosuppres- markers) which accumulate in peripheral blood, bone sive cells, including T regulatory (T-reg) cells, M2 mac- marrow and lymph nodes. A hallmark of the pathophysi- rophages and myeloid-derived suppressor cells (MDSC); ology of CLL is the dysfunction of the immune system (3) depletion and/or inhibition of antitumor immune which is mostly translated in a state of humoral and cellu- cells such as Th1 T-cells and CD8+ cells; and (4) pro- lar immunodeficiency and higher prevalence of autoim- duction of immunosuppressive soluble factors such as mune disorders [2, 3]. IL-10 and TGF-β [4]. These changes are highly relevant for the maintenance and progression of the tumor and contribute to the complex manifestations of the disease. Recently, it has been reported that immune microenvi- *Correspondence: jahernandezr@salud.madrid.org ronment plays a significant role in progression of the dis- 6 C/ Gran Vía del Este 80, 28031 Madrid, Spain ease in contrast to clonal evolution [5]. Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Ab, antibody; BCL-2, B-cell lymphoma 2; BTK, Bruton’s Tyrosine kinase; CAR-T, chimeric antigen receptor T; Fc, fragment crystallizable region; PI3K, phosphoinositide 3-kinase; ROR1, Receptor Tyrosine Kinase Like Orphan Receptor 1; scFv, single-chain variable fragment At the end of the 90´s, the introduction of chemo- should have anti-tumor effects that may help control the therapy in combination with anti-CD20 monoclonal disease. antibodies such as rituximab was an important step There is accumulating evidence of the relevance of the forward in CLL therapy and the outcome of patients interaction between CLL B-cells and the tumor micro- significantly improved. However, most patients will environment. Restoring autologous T-cell responses eventually relapse. Importantly, CIT decreases the dis- remains a potential therapeutic option in CLL, since it ease burden but, at the same time, exacerbates certain should promote anti-tumor immunity without the GvHD defects of an already dysfunctional immune system (graft-versus-host disease) complications of allogenic which further impacts patients’ health, with increased transplants [14]. Here, we review the current treatment infection morbidity and secondary neoplasms [6]. The options (Fig. 1) in CLL in the context of the new molecu- introduction of small molecule inhibitors, such as Bru- lar understanding of the immune system and its interac- ton tyrosine kinase (BTK) [7–10], phosphoinositide tions with this neoplasm. 3-kinase (PI3K) [11], or B-cell Lymphoma 2 (BCL-2) [12, 13], has changed the treatment paradigm by pro- Pathophysiology of the Immune System in CLL viding even more effective therapeutic options for CLL Immune dysfunction is a fundamental characteristic of patients. CLL that can be present even in early stages of the dis- Immunotherapeutic strategies (immune checkpoint ease. However, these immunologic aspects have been a inhibitors, bi-/tri-specific antibodies, chimeric antigen deprioritized side in the research of this field for many receptor (CAR)-T-cells) are emerging as relevant treat- years. The clinical consequences are an increased risk ment options in many malignancies by reverting or of secondary malignancies [15] and infections, the lat- bypassing immunosuppression caused by the tumor, but ter being one of the leading causes of mortality among the results have been disappointing so far in CLL. Effec- patients with CLL [16]. Conversely, in some cases, the tive restoration of the immune competence of a patient perturbation of the immune regulation can result in Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 3 of 16 autoimmune cytopenia in CLL [17, 18]. In this sec- disease progression and possibly involving all immuno- tion, we will review the causes and consequences of the globulin classes. In recent studies, a direct correlation immune dysregulation that characterizes CLL. has been observed between low levels of IgG and IgA and increased mortality due to bacterial infections [6]. Immunosuppressive environment in CLL Regarding T-cell immunity, the absolute numbers CLL B-cells have shown to have immunosuppressive of CD4+ and CD8+ T-cells are higher in patients with effects on normal T and B-cells by direct cell-to-cell con- CLL than in age-matched healthy donors but not propor- tact and by tumor-derived soluble factors [6]. tionally higher since the ratio CD4+/CD8+ is reduced. It has been proposed that CLL B-cells may resem- Interestingly, the reduction of CD4+/CD8+ ratio has ble natural B-reg cells [6] since CLL B-cells also secrete been shown to correlate with disease progression. Also, IL-10, which contributes to their immunosuppressive the impaired function of the main subsets of T-cells has function, and inhibit TNF-α secretion by macrophages. been described including both CD4+ T helper (Th) cells In addition, IL-10 can have autocrine deleterious effects and CD8+ T cytotoxic lymphocytes cells with defects in by promoting proliferation and differentiation of CLL cytoskeletal formation and cytotoxic activity [28]. B-cells. Clinically relevant, increased IL-10 levels have been associated with a diminished survival in CLL patients [19]. Impaired anti‑tumor T‑cell response Moreover, a high number of T-reg cells are observed in The T-cell compartment dysregulation in CLL was CLL, which may negatively impact antitumor immunity described a few decades ago; however, the role of T-cells provided by tumor-infiltrating lymphocytes (TILs) [20]. in the pathogenesis of CLL is still not fully understood. Myeloid-derived suppressor cells (MDSCs) are more CLL B-cell expansion occurs in parallel to an uncon- abundant in CLL patients than in healthy individu- trolled T-cell proliferation, but it is unclear if the increase als. Tumor-associated macrophages (TAMs) secrete is the natural response of the immune system to CLL or if immunosuppressive cytokines (IL-10 and TGF-β). Both it is an active player of the disease. MDSCs and TAMs promote expansion of T-reg cells and Cell to cell communication is also impaired in the inhibit activation of T-cell surveillance providing a sup- CLL environment: synapse dysfunction was identified pressive microenvironment that allows CLL proliferation in T-cells that have been in contact with CLL B-cells [21, 22]. inducing T-cell anergy [29]; PD-1+ T-cell frequency The tumor microenvironment is also immunosup- is increased in progressive CLL which together with pressive in the CLL lymph nodes (LN) with an increased chronic low-affinity self-antigen exposure induces a state number of tumor-supportive lymphocytes (follicular T of “pseudo-exhaustion” [30]. helper (Tfh) and T-reg) and fewer cytotoxic ones, com- Additional perturbations of T-cell subsets have been pared to peripheral blood. CD8+ T-cells, in addition, described and one of the most interesting is the disrup- have an increased expression of the inhibitory recep- tion between Th1 and Th2 balance (Fig. 3). Th1 cells help tors PD-1 that limits their potential to build an immune keep malignant cells under control by the release of mol- response in the long term due to stable epigenetic ecules such as IL-2 and IFN-γ; on the other hand, Th2 changes difficult to revert [23, 24]. cells protect malignant cells by promoting B-cell anti- body production and interfering with cytotoxic T-cells Defects in the innate and adaptive immune systems [31]. In healthy conditions, this system helps maintaining Defects in the innate immune system (Fig. 2) have been normal immune system homeostasis, but it is altered in reported in CLL patients: the phagocytic function and CLL patients. chemotaxis of neutrophils are reduced [25]. Circulat- Naïve CD4+ T-cells differentiation towards Th1, Th2 ing monocytes have gene expression patterns related to or Th17 cells is controlled by interleukine-2-inducible deregulation of phagocytosis and inflammation, poten- kinase (ITK), among other molecules. T-cell receptor tially due to effects of CLL-derived soluble inhibitory (TCR) activation (especially low affinity interactions with molecules [26]. Similarly, NK (natural killer) cells are the major histocompatibility complex (MHC) I) triggers a increased but have impaired production of cytotoxic signal cascade activating ITK and promoting Th2 differ- granules and reduction of cytolytic molecules releases entiation. Th2 produce cytokines such as IL-4, IL-5 and [27]. In addition, low expression of activating molecules IL-13, that trigger positive feedback for proliferation of in the surface of CLL B-cells adds to the already poor Th2 cells and cross-inhibit other Th lineages. In CLL, the function of NK cells [15]. equilibrium is skewed towards Th2 differentiation, inhib- Adaptive immunity is also dysfunctional, with iting effective anti-tumor responses and promoting CLL hypogammaglobulinemia that is exacerbated with B-cell proliferation [32]. Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 4 of 16 Fig. 2 Alterations of the immune system caused by Chronic Lymphocytic Leukemia (CLL) B-cells. BTLA, B- and T-lymphocyte attenuator; CD, cluster of differentiation; CLL, chronic lymphocytic leukemia; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; IFN-γ, interferon gamma; LN, lymph node; MDSC, myeloid-derived suppressor cells; NK, natural killer; TAM, tumor-associated macrophages; PD-1, program cell death protein 1; TCR, T-cell receptor; Th, T helper; TNF-α, tumor necrosis factor alpha; T-reg, regulatory T-cell; X, impairment Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 5 of 16 Fig. 3 Equilibrium between Th1 and Th2 subgroups of T-cells can impact significantly immune tumor surveillance in CLL. CD, cluster of differentiation; IFN-γ, interferon gamma; IL, interleukin; ITK, interleukin-2-inducible T-cell kinase; TCR, T-cell receptor; Th, T helper Recent evidence showed the relevance of another bendamustine/rituximab (BR). For many years, FCR subtype of T-cells important for anti-tumor activity: γδ has been the standard of care in previously untreated T-cells can identify and kill tumor cells in a MHC-inde- young and fit CLL patients. FCR resulted in high over- pendent manner and recognize a broader spectrum of all response rates with a complete responses of 44%, and neoplastic antigens, but these cells are also affected by median progression-free survival (PFS) of 51.8 months the immunosuppressive environment of CLL [33]. [36]; the benefit was primarily driven by low-risk patients with mutated IGHV and without 17p deletion [37]. How- Current treatment approaches and impact ever, this combination is considered too toxic for frail in the immune function and/or elderly patients due to appearance of hematologi- Chemoimmunotherapy cal toxicities [36]. Fludarabine can cause reductions in Purine analogs such as fludarabine, cladribine, or pen- T-cell numbers, with a higher effect on CD4+ cells [34], tostatin, and alkylating agents such as bendamustine, γδ [34] and T-reg cells [38], and serious opportunistic chlorambucil or cyclophosphamide, interfere with both T-lymphopenia–associated infections during the first tumor and healthy dividing cells. As a consequence, year after FCR treatment, such as Pneumocystis jirovecii chemotherapy reduces CLL B-cell numbers, but also and Legionella pneumoniae, pulmonary aspergillosis, decreases other healthy cells including T-cells and slows endemic fungus infections or disseminated listeriosis down the recovery to normal levels, which ultimately [39]. Also, long-term toxicities including risk of second- leads to infections [34, 35]. ary hematological malignancies (i.e, acute myeloid leu- Two frequently used CLL regimens until recently are kemia, myelodysplasia) have been associated with the fludarabine/cyclophosphamide/rituximab (FCR) and FCR regimen [40]. T-cell repertoire renewal after FCR Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 6 of 16 treatment was found to be affected through ablation and changes in the tumor environment due to a decrease in immune reconstitution, rather than expansion of previ- the expression of immunosuppressive molecules such as ous T-cell clones, thus impairing early antigen recogni- PD-L1, IL-10, CD200 or BTLA in CLL B-cells [49, 50]. tion [41]. Interestingly, another related Tec kinase, ITK, is also BR has demonstrated efficacy in CLL although not as inhibited by some of these small molecules. ITK is an significant as FCR [42]. This regimen is frequently used in important component of TCR signaling and promotes patients unsuitable for fludarabine-based treatments. It is Th2, T-reg, and Th9 CD4+ cells differentiation and associated with fewer hematological complications and reduces cytotoxic CD8+ and CD4+ Th1 cells differentia- infections compared FCR, but reductions of CD4+ cells, tion [31]. This is relevant for tumor control since skew- delays in recovering normal CD4+ cells levels and infec- ing T-cell differentiation towards a Th2 response would tions were common [35]. create a pro-tumor environment and cause a decrease of Anti-CD20 antibodies (rituximab and obinutu- Th1 functions in tumor surveillance. Therefore, the inhi- zumab) target CLL B-cells for complement-dependent bition of ITK also has an impact on the T-cell compart- cytotoxicity (CDC), direct cell death, antibody- ment [31]. dependent cell-mediated phagocytosis (ADCP) or Ibrutinib elicits changes in the tumor microenviron- antibody-dependent cellular cytotoxicity (ADCC) by ment that both control CLL B-cells proliferation and NK cells. Therefore, even though CLL B-cells express reestablish immune surveillance (Fig. 4). Regarding the low levels of the necessary ligands to activate NK cyto- T-cell compartment, ibrutinib causes a transient increase lytic activity, anti-CD20-coated CLL cells are able to of CD4+ and CD8+ T-cells numbers, mostly effector trigger an ADCC response [15]. It has been reported and effector memory T-cells [50], and is associated with that rituximab treatment may reduce humoral an expansion in the TCR repertoire diversity [51]. In response to “recall antigens”, which may be due to the the long term, ibrutinib causes a decrease in pathologi- depletion of healthy antibody-producing B-cell clones, cally high circulating B-cells and exhausted and chroni- although how this may affect the rate of infections or cally activated T-cells but preserved naïve T-cells and NK secondary malignancies is not clear yet [43]. cells [52]. Ibrutinib also shifts the equilibrium of CD4+ Obinutuzumab, a type II humanized anti-CD20, has T-cells from Th2 and Th17 cells to a Th1 environment by shown different properties from rituximab, displaying inhibiting ITK, reverses CD8+ cell exhaustion [53] and superior ADCC and direct cell death activity, simi- favors activation of CD8+ cytotoxic T-cells [31]. Moreo- lar ADCP but inferior CDC in vitro. Obinutuzumab ver, it has been observed that ibrutinib-based therapy caused significant and sustained reductions of CD4+ improves functional immune and cytolytic synapses cells after one infusion. In addition, a reduction of between T-cells and CLL B-cells compared to FCR treat- CD8+ cells was also detected but levels were normal- ment, and enhances the polarization of perforin towards ized after 6 months [44]. These effects were observed CLL B-cells in T-cell synapses [41], helping to restore the 24-72h post-infusion while depletion of B-cells suf- cytotoxic capacity of T-cells to kill tumor cells [54]. fered no significant reduction until later. One more In addition, ibrutinib is associated with a the reduction effect in the immune system was the early and sus- of the T-reg/CD4+ cells ratio, the net increase of Th17 tained reduction in NK cells [44]. cells and the inhibition of the expression of PD-1 and Therefore, immune-related risks associated with CTLA-4 in CD4+ and CD8+ T-cells all together leading anti-CD20 therapy are infections, for example, hepati- to a reduction in the immunosuppressive status and an tis B reactivation, which has been described in patients improvement in immune surveillance [50, 55]. with CLL and other B-cell lymphomas [45]. Ibrutinib also modulates the function of other immune cells, such as macrophages, by reducing the cell-cell con- Novel Agents tacts with CLL B-cells in the bone marrow, migration and BTK inhibitors nitric oxide production [55, 56], and of monocytes, by BTK belongs to the Tec family of kinases and plays a role impairing differentiation towards M1 macrophages, caus- in immune cell signaling. BTK is part of the BCR (B-cell ing adhesion and phagocytic deficiencies and increasing receptor) signaling pathway that promotes survival and the immunosuppressive profile of nurse-like cells [57]. proliferation of malignant B-cells. BTK inhibitors, includ- These changes in the immune system triggered by the ing ibrutinib, acalabrutinib and zanubrutinib have shown treatment with ibrutinib have positive clinical relevance; clinical efficacy in patients with CLL [7, 46–48]. for example, the restoration of TCR diversity has been BTK inhibitors produce transient lymphocytosis shown to correlate with clinical efficacy and lower infec- caused by the mobilization of B-cells from LN, bone mar- tion rates [31, 51, 53]; and the restoration of the CD4+/ row and spleen to peripheral blood. They also produce Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 7 of 16 Fig. 4 Effects of ibrutinib on immune cells and tumor microenvironment. BTLA, B- and T-lymphocyte attenuator; IL-10, interleukin 10; NAMPT, Nicotinamide Phosphoribosyltransferase; PD-L1, programmed cell death protein ligand 1; TCR, T-cell receptor CD8+ ratio may help improve pre-existing autoimmune Zanubrutinib is another highly specific BTK inhibitor cytopenias and decrease the rate of emergent events [18]. with low ITK activity that has shown potential in CLL In summary, ibrutinib effectively controls CLL via a [48]. It has shown significant reductions on PD-1 and dual mechanism. First, it has shown to act directly on CTLA-4 expression and a decrease in T-reg cells; but no CLL B-cells, reducing the number of malignant cells and changes in Th1/Th2 ratio [60]. their immunosuppressive effects and second, through modulation of the microenvironment and restoration of Pi3K inhibitors the physiological immune function, by increasing T-cell- PI3K-δ is mostly expressed in hematopoietic cells and dependent immune surveillance. With the available evi- frequently overexpressed in B-cell lymphomas. It plays a dence up to date, ibrutinib is the first therapy to show major role in B-cell signaling and has also been involved that a strategy to address tumor burden and immune in T-reg cells and MDSC function [11]. Two Pi3K inhibi- function simultaneously could improve the disease man- tors have been approved, idelalisib and duvelisib). Idela- agement (Fig. 4). Moreover, this offers significant poten- lisib is a PI3K-δ selective inhibitor with efficacy in CLL tial to combine with other therapeutic options. by inhibiting CLL B-cell proliferation, survival, adhesion, Acalabrutinib is a selective inhibitor of BTK that has and homing [11]. shown efficacy in CLL [46, 58]. Since ITK binding is Idelalisib has also effects on non-B-cells: it decreases essentially absent for acalabrutinib, the effects on Th2 the number of T-reg cells, although this effect was to Th1 switch in the CD4+ T-cell compartment are observed mostly in patients with hepatotoxicity [61], and not observed [50]. Similarly to ibrutinib, a reduction of impairs their differentiation and suppressive functions immunosuppressive molecules such as CD200, BTLA, [20]. In addition, it decreases the production of cytokines PD-1 and CTLA-4 expression, together with a decreased and other factors such as TNF-α, CD40L and IL-6 by production of IL-10, have been noted [50]. Acalabru- T-cells; IFN-γ by NK cells and IL-10 by T-reg cells [20]. tinib has some other effects on the immune system, for The reduction of T-reg cells and soluble pro-tumor fac- example, it impairs efficient responses on macrophages tors makes idelalisib an interesting candidate for com- (M1 polarization and TNF-α secretion) and neutrophils bination with immunotherapies but it is not frequently (phagocytosis, oxidative burst and others), important for considered an option because those additional effects antimicrobial infection control [59]. Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 8 of 16 have been associated with relevant immune-related tox- Richter transformation (RT), and limited in CLL without icities of concerning severity and frequency, such as RT [67]. Some studies have shown that addition of ibru- hepatotoxicity or enterocolitis [61]. tinib to the therapy with ICI may improve its efficacy, especially in patients with RT [68, 69]. Another strategy arising in oncology is the develop- BCL‑2 inhibitors ment of Bi/Tri-specific antibodies. These molecules are BCL-2 is an anti-apoptotic factor that is overexpressed able to force the interaction between tumor and cytotoxic in CLL. Venetoclax is a BCL-2-selective BH3-mimetic cells and their domains can be combined to target dif- that has been effective in treating patients with CLL, by ferent molecules, for example, CD19, CD20 or ROR1, in directly inducing apoptosis of CLL B-cells [12]. B-cells and CD3 in effector T-cells, although other com- Venetoclax has also effects on the T-cell compart- binations and different effector cells (NK cells) have been ment, reducing the total number of CD4+ and CD8+ used as well [2]. cells although this is thought to be an indirect effect of CD19/CD3-scFv-Fc bi-specific antibodies are able the decrease of the tumor. Among the surviving cells, to recruit autologous T-cells to kill malignant cells and there was a high proportion of CD4+ and CD8+ effec- have shown potential in treating CLL. For example, it tor memory T-cells while the proportion of naïve T-cells has been observed that blinatumomab is able to medi- was lower, these remaining cells kept their proliferation ate CLL B-cells and cytotoxic T-cells interaction and capacity intact [62]. In addition, venetoclax reduces the trigger tumor cell death in vitro but no activity has been immunosuppressive environment by reducing the num- detected in vivo [70]. Interestingly, these effects may ber of Tfh cells, T-reg cells and PD-1+ CD8+ T-cells, as be faster in samples of patients treated with some BTK well as the overproduction of inflammatory cytokines inhibitors [71]. Similarly, ROR1xCD3 bi-specific antibod- [62]. ies had cytotoxic activity against CLL B-cells, but when Moreover, some studies suggest venetoclax reduces the autologous T-cells were used, only those isolated from number of NK cells but restoration of their function has patients previously treated with ibrutinib had a signifi- been observed after treatment [63]. cant effect [72]. Immunotherapies Effects of treatment combinations on the CLL CAR-T cells are T-cells from a patient that are modified immune environment ex vivo and reintroduced to target cancer cells. Infusion Considering the potential of the different therapies dis- of ex-vivo manipulated cells may reduce the influence of cussed above on the restoration of the T-cell compart- the immunosuppressive tumor microenvironment and, ment and other components of the immune system, it conversely, the use of patient’s own cells should reduce is reasonable to try different combinations that, at least the risk of GvHD [14]. Antigens of choice for initial theoretically, could improve the patient outcomes by CAR-T cells were the surface antigens CD19, CD20 and leveraging the patient’s own immune system response CD23, causing also a reduction in healthy B-cells. Over- (Table 1). all, either because of lack of efficacy or the development of adverse events, CAR-T cells have not reached clinical New agents + anti‑CD20 antibodies combinations expectations in CLL [64, 65], at least partially due to the BTK inhibitors have been studied in combination with innate dysfunction in T-cells coming from patients from anti-CD20 antibodies. BTK inhibitors can inhibit anti- which the CAR-T cells are produced. Interestingly, thera- CD20-induced NK cell cytokine secretion, cell degran- pies that restore immune competency, such as ibrutinib, ulation and NK ADCC in vitro [73] but still, these may enhance the efficacy of CAR-T cells [63]. A similar combinations have demonstrated preserved activity of approach is being studied with CAR-NK cells, and pre- anti-CD20 antibodies clinically. In fact, similar or supe- liminary results suggest better results may be obtained in rior PFS has been observed between acalabrutinib + CLL [66]. obinutuzumab vs acalabrutinib alone [58]. Other studies Immune Checkpoint Inhibitors (ICI) reactivate have shown positive synergistic effects when ibrutinib is immune response towards tumors by blocking inhibitory combined with obinutuzumab [8]. signals mediated by PD-1 and CTLA-4. ICI have trans- Impact of idelalisib combinations in CLL and specifi- formed the treatment paradigm in many solid tumors, cally in the T-cell compartment has been studied to a but their efficacy remains suboptimal in CLL. PD-(L)1 lesser extent. Idelalisib has similar effects to ibrutinib pathway is active in CLL but the expression of PD-L1 in when tested in vitro in combination with anti-CD20 CLL B-cells is usually low. In recent trials, the efficacy of antibodies, although inhibition of antibody-dependent anti-PD-1 monotherapy has been moderate in CLL with Table 1 Available evidence of effects of different therapeutic agents alone or in combination on the immune system in chronic lymphoid leukemia (CLL). CIT BTKi Pi3Ki BCL2i ImmTher CIT • FCR: T-cells decrease, mostly CD4+, • BTK inhibitors can inhibit anti-CD20- • Ide may marginally reduce anti- • V + Obi: reduction in healthy B-cells, γδ and T-reg induced NK cell cytokine secretion, CD20 effects T-cells (Tfh, T-reg and PD-1+ CD8+ • BR: T-cells decrease (CD4+) cell degranulation and FcR-stimulated T subtypes) and NK cells. Decrease in • C: T-cells decrease NK ADCC in vitro IFN-γ and TNF-α produced by CD8+ • Anti-CD20: complement pathway • Ibr + anti-CD20: Enhancement of T-cells. Improved NK cell function exhaustion, reduction of humoral ADCP response, reduction of CD4+ and Moreno et al. J Exp Clin Cancer Res transient reduction of CD8+ T-cells, NK cells decrease BTKi • Ibr, Aca, Zanu: Decrease of IL-10, • Ibr+V: reduction in healthy B-cells, • Ibr + CAR-T cells: Improved efficacy of CD200 or BTLA in CLL B-cells. Reduce T-cells (Tfh, T-reg and PD-1+ CD8+ CAR-T cells in ibr-treated patients macrophage function, neutrophil to T subtypes) and NK cells. Decrease • Ibr + bi-specific antibodies: enhanced macrophage differentiation. Reduc‑ in IL-4 by CD4+ cells and TNF-α activity in Ibr-treated patients (2021) 40:321 tion of PD-1 and CTLA-4 expression produced by CD8+ T-cells. Trend • Ibr + ICI: Improved response to anti- in T-cells of improvement in the antibody PD-L1 treatment, especially patients • Ibr: Transient increase of effector production with RT T-cells. Increase in TCR diversity and • br may improve autologous Vγ9Vδ2 Th1 cells, activation of CD8+ T-cells. T-cell therapy Reduction of exhausted and chroni‑ cally activated T-cells. Preservation of naïve T-cells and naïve NK cells. Decrease of Th2 and T-reg cells. Improved immune and cytolytic syn‑ apses between T and CLL B-cells. Pi3Ki • Ide: Decrease of T-reg cells, impairs • Ide-treated patients showed improved their differentiation and suppressive autologous CAR-T cells generation, functions. Decreases TNF-α, CD40L expansion and cytotoxic effects. and IL-6 by T-cells; IFN-γ by NK cells • Idelalisib caused a decrease in expres‑ and IL-10 by T-reg cells sion of PD-1 BCL2i • V: reduction of CD4+ and CD8+ cells, increase proportion of effector memory T-cells vs naïve T cells, reduction of Tfh, T-reg and PD1+ CD8+ T-cells. Decreased NK cells but function restored ImmTher • CAR-T cells can restore functional capacity of T-cells by in vitro modifica‑ tion • Bi/Tri-specific antibodies may improve interactions between cytotoxic T-cells and CLL B-cells Aca, acalabrutinib; ADCC: antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cell-mediated phagocytosis; BCL-2i, B-cell lymphoma 2 inhibitor; BR: bendamustine plus rituximab; BTKi, Bruton’s Tyrosine kinase inhibitor; BTLA, B- and T-lymphocyte attenuator; C, cyclophosphamide; CAR, chimeric antigen receptor; CIT: chemoimmunotherapy; CLL, chronic lymphocytic leukemia; FcR, receptor of antibody fragment crystallizable region; FCR: fludarabine, cyclophosphamide, rituximab; Ibr: ibrutinib; ICI, immune checkpoint inhibitor; Ide: idelalisib; IL, interleukin; ImmTher, immunotherapies; NK, natural killer; Obi: obinutuzumab; PD-1, programmed cell death protein 1; PD-L1, programmed cell death protein ligand 1; Pi3Ki, phosphoinositide 3-kinase inhibitor; T-reg, regulatory T-cell; TCR, T-cell receptor; Tfh, follicular T helper; TNF, tumor necrosis factor; RT, Richter transformation; V, venetoclax; Zanu, zanubrutinib. Page 9 of 16 Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 10 of 16 cell-mediated effector mechanisms seems to be less pro- that co-administration of ibrutinib and CD19-directed nounced [73]. CAR-T cells (CTL019) in a CLL mouse model improves Studies combining venetoclax and obinutuzumab have the efficacy of the CAR-T cells. Ibrutinib can improve the shown a decrease not only in CLL B-cells but also healthy proliferative capacity of T-cells from CLL patients, and B-cells, T-cells and NK cells. Decreases seem to be more it has been shown that the critical expansion of CTL019 profound in Tfh, T-reg and PD-1+ CD8+ T subtypes. In is greater when collected from patients that have under- addition, this combination generates a decrease in IFN-γ gone at least 5 cycles of ibrutinib treatment. In addition, and TNF-α produced by CD8+ T-cells [23]. In clinical PD-1 and CD200 inhibitory molecules decrease expres- trials, venetoclax plus anti-CD20 antibodies combina- sion in T and CLL B-cells, respectively. Interestingly, tions have shown durable responses, in some cases even these effects have not been observed when cells are after cessation of treatment, although the effects of these treated in vitro with ibrutinib [63]. In clinical trials, ibru- combinations on the immune function need to be further tinib-treated patients had significantly better response investigated [74]. rates to CAR-T cells compared to ibrutinib-naïve ones, with morphologic complete responses, measurable resid- New agents + Immunotherapy ual disease negativity, absence of disease in lymph nodes Numerous reports have shown that ibrutinib enhances and less severity in cytokine release syndrome [65, 76]. the effects of immunotherapies. It has been hypoth- Another type of cell therapy involving another sub- esized [75] that ibrutinib could restore antitumor T-cell population of T-cells, Vγ9Vδ2-T-cells, has shown poten- immune response in an ITK-dependent manner and tial in CLL. These cells are able to recognize metabolites enhance ICI effectiveness. A lymphoma model with an produced during malignant transformation and trigger ibrutinib-insensitive BTK version has been developed a MHC-independent cytotoxic response [33]. Similar and it has been observed that anti-PD-L1 treatment is to other T-cells, Vγ9Vδ2-T-cells function is impaired in T-cell-dependent and moderately effective but the com- CLL, and cell expansion in vitro is inefficient. Informa- bination with ibrutinib led to significant improvement tion regarding this subpopulation of T-cells is limited but in the response, through its effects in the immune func- preliminary results suggest that ibrutinib may induce Th1 tion restoration rather than the direct effects in CLL skewing of these cells in vitro, making it a potential com- B-cells [75]. Two clinical trials have reported data on the bination strategy for autologous Vγ9Vδ2 T-cell therapy combination of nivolumab and ibrutinib. Activity of the [33]. combination is limited in CLL patients but is promising Idelalisib has been shown to improve autologous in patients with RT [67, 69]. An additional clinical trial CAR-T cell generation, expansion and cytotoxic effects reported results on the use of pembrolizumab in CLL when isolated from idelalisib-treated patients or when and RT patients, where 4 out of 9 patients with RT had incubated with idelalisib. It promotes enrichment of objective responses, and none of the 16 patients with naïve-like T-cells and increase of CD8+ cytotoxic T-cells. CLL. Interestingly, all 4 patients achieving response were It has also been observed that idelalisib causes a decrease previously treated with ibrutinib [68]. in expression of PD-1 [77]. An explanation of these results may be that CLL B-cells BTK inhibitors have also demonstrated enhancement and RT-cells may be expressing different tumor neoanti- of bi-specific antibody efficacy as previously explained gens (or RT-cells may be more efficient presenting them). creating an interesting rationale to combine these agents. This causes that only RT-cells neoantigens are identified by T-cells, helping ICI trigger responses against them, Other combinations executed by ibrutinib-mediated re-activated T-cells [67]. Both ibrutinib and venetoclax have been reported to Venetoclax helps keep effector memory T-cells viable reduce tumor pro-survival Tfh and T-reg cells and re- and functional, and this may be important to improve the activate T-cells as monotherapies [50, 78]. The combina- effects of immunotherapies [62], although a decrease in tion of both molecules has been studied in patients with naïve T-cells may reduce the response to tumor-derived CLL with results reaching superior complete responses neoantigens. So far, there is only preliminary in vitro evi- and MRD negativity [79]. Ibrutinib enhances venetoclax dence that venetoclax may have synergistic effects with killing effects by sensitizing CLL B-cells to the BCL-2 ICI and therefore further research is needed to confirm antagonism of venetoclax [80]. this observations [62]. Regarding effects in the immune environment, research is still ongoing but there is some data from the combina- New agents + cell therapies tion of venetoclax with ibrutinib or obinutuzumab [23]. There is also evidence that ibrutinib can help enhance With both venetoclax plus ibrutinib or venetoclax plus the CAR-T cell therapy. A preclinical study showed obinutuzumab strategies, reductions in healthy B-cells, Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 11 of 16 NK cells, and T-cells, specifically a reduction of Tfh- resistance beyond classic genetics. Single-cell technolo- cells, T-reg cells and PD-1+ CD8+ T-cells are observed gies, immune-phenotyping, single-cell transcriptome in vitro [23], mediated mostly by venetoclax. profiling, functional studies, and chromatin mapping, A decrease in the production of IFN-γ and TNF-α in all integrated with bioinformatic analytic tools, may help CD8+ T-cells has only been observed in the venetoclax understand the supportive role of the immune microen- plus obinutuzumab combination while a decrease in IL-4 vironment in CLL progression and the co-evolution of production by CD4+ T-cells has only been observed in cells involved in the disease, directly or indirectly [5, 41, the venetoclax plus ibrutinib combination, consistent 52, 82–84]. with the restoration of the Th1/Th2 balance described Mutations in BTK and PLCG2, the kinase immedi- for ibrutinib [23]. Other relevant differences are that an ately downstream of BTK, have been described as com- improvement in NK cell function has only been observed mon mutations in patients progressing on ibrutinib [85, in the venetoclax plus obinutuzumab combination while 86]. Interestingly, these resistant clones would provide a trend of improvement in the antibody production has protection to neighboring cells with wildtype BTK [87], only observed in the venetoclax plus ibrutinib combina- and they were detectable many months before clinical tion [23]. progression [88]. Resistance to ibrutinib can go beyond CLL is a heterogeneous disease caused by dysregula- the BTK pathway, and it has been described for MCL that tion at multiple levels. Current treatment options are not tumor microenvironment provides initial drug resistance curative and complex combinations with multiple drugs through interactions with the malignant cells through are being tested to reduce the residual disease in a dura- integrin β1-ILK [89] and through multiple other signal- ble way and improve anti-tumor response by the immune ing networks in CLL, identified by patient- specific signa- system. Targeting various disrupted pathways at the same tures [83]. time seems a reasonable option to increase the efficacy Similarly, mutations in BCL2, detectable months ear- and reduce relapses, but careful consideration must be lier than clinical progression using droplet digital PCR, taken because the molecular pathways leading to disease have been shown to decrease the efficacy of venetoclax and the off-targets effects are not fully understood. Key [90]. Activated T-cells produce IL-4 and IL-21 that can factors to consider when designing combination studies stimulate the CD40-CD40L interaction and increase are (1) targeting different molecular pathways with scien- the expression of the anti-apoptotic factor BCL-xL, tific evidence of synergistic effects, (2) using treatments among other factors [91]. with non-overlapping safety profiles, (3) designing the One of the most frequently used strategies to avoid treatment regimen to minimize undesired effects and (4) proliferation of resistant clones is combining different including a significant set of biomarkers that allows the targeted therapies. Combination of ibrutinib and vene- interpretation of the results. toclax would reduce the probabilities of proliferation Examples of such complex regimes are ibrutinib plus of clones with resistant mutations in both pathways obinutuzumab plus venetoclax, designed to maximize the simultaneously. Additionally, it has been shown that efficacy keeping adverse events low. Positive results have microenvironment cytokines (IL-10, sCD40L or CpG- been recently confirmed in a phase 2 trial [81], showing ODNs) can significantly reduce the synergy of the com- deep responses, and an acceptable safety profile. Various bination in an NF-κB-dependent manner [91, 92]. phase 3 cooperative group studies are currently ongo- In general, the immune microenvironment creates ing (NCT04608318, NCT03701282 and NCT03737981). protective conditions through a network of complex Other combinations still need further investigation. signal interactions to resist therapeutic interventions. Hypoxia has been identified as key condition on set- ting a protective niche for CLL cells. Under hypoxia, Role of the immune system in resistance macrophages acquire a tumor-permissive M2 pheno- to treatment type, T-cells become immunosuppressive and MDSC Despite recent progress on treatments for patients with increase presence [93, 94] directly impacting cell sur- CLL, progressive disease is still frequent in many cases. vival and drug resistance. Ibrutinib-induced apoptosis, Due to selective pressure in the presence of therapies, for example, is reduced under these conditions [95], resistant clones may proliferate and cause the disease but at the same time, ibrutinib treatment can coun- to progress. Targeted therapies are especially prone to teract, at least partially, the establishment of the pro- resistance with relatively simple mutations in the tar- tective microenvironment, by suppression of NAMPT get protein or pathway, but thanks to methodologi- (factor involved in linking oxygen use, metabolism, and cal advances, we are starting to understand much more immune function) transcription, important for the dif- complex signaling networks that can create forms of ferentiation of tumor-supporting M2 macrophages Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 12 of 16 [96]. A key mediator of the hypoxia effects is HIF-1, immunoglobulin levels [98] and a reduction of infec- found to be overexpressed in CLL [95] and it has been tions has been reported when ibrutinib is used beyond 6 shown that inhibition of HIF-1 with BAY87-2243 has months [99]. Moreover, ibrutinib may also attenuate the synergistic effects when combined with other agents, exacerbated immune response in severe infections, such such as ibrutinib [95]. as in the case of visceral leishmaniasis [100] or Staphylo- Interestingly, hypothesis-generating studies have coccus aureus [101], or reduce the burden of the inflam- shown that impairing the microenvironment can matory process during the infection, for example in the help improve efficacy of targeted therapies in general. case of COVID-19 [102] or pneumococcal pneumonia Ascorbic acid is a pro-oxidant agent that has shown to [103]. overcome the protective effect of the microenviron- ment. It can potentiate the effects of ibrutinib, idelal- isib and venetoclax in vitro and it was able to reduce Autoimmune cytopenias the viability of CD40L/IL-4- stimulated CLL cells [97]. Chemoimmunotherapy may impair further the func- Even though these results are preliminary, new high tion of effector T-cells, and in some cases, such as throughput and single-cell methods may help under- fludarabine-based regimens, may increase the risk of stand better the complexity of these interactions and autoimmune cytopenias (AIC), although addition of can contribute to design therapeutic combination cyclophosphamide appears to decrease the incidence of strategies that target the tumor microenvironment to fludarabine-dependent AIC [17]. Idelalisib has shown reduce progression in CLL and identify patterns that to reduce T-reg cells, which are key players in the con- are able to predict response to treatment. trol of autoimmune responses and excessive reactions to non-self-antigens [61]. In the case of ibrutinib, data have Clinical considerations of immune restoration/ shown that it causes a reduction of Th17 cells, decreasing Expert insight the risk of treatment-emergent AICs [17], and providing Patients with CLL have a characteristic disruption of better control in patients with pre-existing AIC [18]. the immune function and addressing it in parallel to the tumor burden should be central in the management of Secondary malignancies the disease. Treating the disease by restoring the anti- The incidence of secondary malignancies is increased in tumor activity of the patient’s immune system can lead to CLL patients potentially due to the disrupted immune sustained and deep responses and reduce side effects and surveillance. CIT regimens such as FCR have been asso- non-progression deaths. ciated with a higher incidence of secondary malignancies, CLL treatments may exacerbate immune defects, caus- particularly hematological neoplasias [40]. This effect has ing infections, autoimmune diseases or secondary neo- been shown to be exacerbated in patients with prolonged plasias [98]. Data on these effects are scarce but we are cytopenias, potentially due to the prolonged treatment starting to understand the impact of CLL treatments on with rituximab. Recently, in a retrospective unicentric these side effects: study of patients treated with BTKi in first-line (20% of patients) and in the relapse/refractory setting (60% of patients received alkylating agents), the observed rate of Infections secondary malignancies was 2.2, similar to the reported Chemoimmunotherapy effects on the T-cell compart- rate in a large cohort of CLL patients followed at a single ment may also lead to infections [16]. Idelalisib has been tertiary center prior to the use of BTKi [104–106]. In the associated with an increase in the risk of Pneumocystis CLL14 trial, secondary malignancies seem more frequent jirovecii pneumonia and other opportunistic infections in venetoclax combined with obinutuzumab (18%) vs. [61]. ICIs have shown multiple examples of immune- chlorambucil plus obinutuzumab (10.3%) after a follow- related adverse events in different tumors, requiring up of 3 years. Additional follow-up will be needed to con- immunosuppressive treatment leading to secondary firm this association [13, 107]. infections [98]. Blinatumomab causes hypogammaglobu- linemia and venetoclax has been associated with neutro- Conclusions penia and infections [98]. A slight increase in bacterial, CLL has undergone relevant clinical advances in the past viral and opportunistic infections has been observed decades, both in the understanding and the treatment of with ibrutinib, caused by defects in the innate immune the disease. For many years, the focus of the therapeutic function, although these infections appear mostly dur- approaches has been to decrease the number of B-cells to ing the first months of therapy. Interestingly, prolonged reduce the tumor burden. New insights have shown that treatment with ibrutinib restores humoral immunity and the interaction of leukemic cells with T-cells and other Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 13 of 16 players of the immune system participate in the patho- practice until a better understanding of the disease and genesis of the disease and contribute to a decrease of the the effects of the different treatments is achieved. overall health status of the patient, but this knowledge There is still need for more research to understand the has not fully been applied to clinical practice yet. Some effects of each treatment option (monotherapies and real-world studies are starting to look at relevant immune combinations) in the tumor environment and current parameters, such as IgA in ibrutinib-treated patients technology can yield insightful data. Thus, it is essential [108] but more information is needed to have a broader to include broader biomarker analysis in clinical studies, picture of the dynamics of the whole immune system in that go beyond analysis of B-cells and include T-cell sub- the treated patients. We need to further understand the populations and soluble factors at baseline and different roles of the different immune cells in the disease to apply timepoints. therapeutic strategies that not only eliminate malignant Moreover, whatever we learn about immunothera- B-cells but also restore the immune competence of the pies in such a paradigmatic immunosuppressed disease patient toward anti-tumor surveillance and immunity. as CLL can help improve their efficacy in other tumors There is growing evidence that T-cells play a very rel- where immune function is impaired. evant role in the disease progression and understand- ing how T- and B-cells create a pro-tumor environment Abbreviations is essential to treat CLL patients. CLL B-cells can cause ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent an immunosuppressive effect on T-cells, which exhibit cell-mediated phagocytosis; AIC: autoimmune cytopenias; BCL-2: B-cell Lym‑ exhaustion features that abrogate immune control of the phoma 2; BCR: B-cell receptor; BR: bendamustine/rituximab; BTK: Bruton tyros‑ ine kinase; CAR​: chimeric antigen receptor; CDC: complement-dependent tumor. A whole pro-tumor strategy is put in place by the cytotoxicity; CIT: Chemo(immuno)therapies; CLL: Chronic Lymphocytic Leu‑ immune system: decrease in Th1 cells (anti-tumor) is kemia; CpG-ODNs: CpG oligodeoxynucleotides; CTL019: CD19-directed CAR-T offset by an increase of Th2 cells (pro-tumor), cytotoxic cells; FCR: fludarabine/cyclophosphamide/rituximab; GvHD: graft-versus-host disease; HCK: hematopoietic cell kinase; ICI: Immune Checkpoint Inhibitors; T-cells number and function are inhibited, immunosup- ILK: integrin-linked kinase; ITK: interleukine-2-inducible kinase; LN: lymph pressive MDSC, T-reg cells and Tfh cells increase their node; MCL: mantle cell lymphoma; MDSC: myeloid-derived suppressor cells; function, inhibitory cytokines (i.e. IL-10) and checkpoint MHC: major histocompatibility complex; NAMPT: nicotinamide phosphoribo‑ syltransferase; NF-κB: nuclear factor kappa-light-chain-enhancer of activated receptors (i.e. PD-1) are upregulated and the conditions B cells; NK: natural killer; PFS: progression-free survival; PI3K: phosphoinositide in the lymph nodes are even more immunosuppressive. 3-kinase; PLCG2: phospholipase C gamma 2; RT: Richter transformation; T-reg: Keeping in mind that not only eliminating malignant T regulatory cells; TAM: Tumor-associated macrophages; TCR​: T-cell receptor; Tfh: follicular T helper; Th: T helper; TILs: tumor-infiltrating lymphocytes. B-cells, but also restoring the immune function may have significant positive consequences in the clinical prac- Acknowledgements tice, especially in CLL, that immune dysfunction is the The authors wish to thank Alberto Moldón, PhD for editorial support. cause of most of the complications for this disease. Also, Authors’ contributions understanding the relevance of the other immune players All the authors defined the general structure and content of the manuscript. in CLL and how different treatments affect them would Dr. Hernández-Rivas and Dr. Villanueva drafted the first version of the article, and Dr. Moreno, Dr. Muñoz and Dr. Terol revised the manuscript and provided have a direct impact in successfully managing the disease. additional information. All the authors have read and approved the final First, because this will allow us to avoid therapies that manuscript. negatively impact the immune function, such as some Funding chemotherapies. Second, because it will reduce the num- Janssen-Cilag, S.A. has provided financial aid for editorial and publication fees ber of adverse events related to the immune dysfunction, for example infections, autoimmunity and second malig- Availability of data and materials Not applicable nancies. Third, because this will facilitate the design of combination strategies, not only to reach deeper durable Declarations responses but to attack the disease from different fronts, with higher chances of synergistic and long-term effects. Ethics approval and consent to participate And four, it will allow us to successfully use the new Not applicable generation of therapeutic strategies, most importantly Consent for publication immunotherapies and cell therapies, in CLL, so far inef- Not applicable ficacious due to the immune dysfunction in CLL patients. Competing interests Management of CLL multilaterally may be the best Carol Moreno is a consultant for Abbvie, Acerta, AstraZeneca, Janssen, option for patients but still, caution with unexpected Pharmacyclics and Sunesis; received speakers bureau fees from Abbvie and effects, and prevention of known adverse events together Janssen; and received grant/research support from Gilead, Janssen and Roche. Cecilia Muñoz is a consultant for Janssen. María José Terol is a consultant for with prompt management of side effects may help in the Abbvie, Gilead, Janssen, Roche and Takeda; received grant/research support from Gilead and Janssen. José-Ángel Hernández-Rivas is a consultant for Moreno et al. J Exp Clin Cancer Res (2021) 40:321 Page 14 of 16 Abbvie, Amgen, AstraZeneca, Beigene, Celgene, Gilead, Janssen, Roche, Rovi 16. Morrison VA. Infectious complications of chronic lymphocytic leukae‑ and Takeda; received speakers bureau fees from Abbvie, Amgen, AstraZeneca, mia: Pathogenesis, spectrum of infection, preventive approaches. Best Celgene, Gilead, Janssen, Roche and Takeda; and received grant/research sup‑ Pract. Res. Clin. Haematol. 2010:145–53. port from Celgene. Miguel Villanueva is an employee of Janssen-Cilag, S.A. 17. Rogers KA, Ruppert AS, Bingman A, Andritsos LA, Awan FT, Blum KA, et al. Incidence and description of autoimmune cytopenias during Author details treatment with ibrutinib for chronic lymphocytic leukemia. Leukemia. 1 Hospital Santa Creu i San Pau, Barcelona, Spain. 2 Hospital Universitario de la 2016;30:346–50. Princesa, Madrid, Spain. 3 Hospital Clínico de Valencia, Valencia, Spain. 4 Hospital 18. Vitale C, Ahn IE, Sivina M, Ferrajoli A, Wierda WG, Estrov Z, et al. 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