Dynamic changes in HLA-DR expression during short-term and long-term ibrutinib treatment in patients with chronic lymphocytic leukemia

Accepted Manuscript Title: Dynamic changes in HLA-DR expression during short-term and long-term ibrutinib treatment in patients with chronic lymphocytic leukemia Authors: Gayane Manukyan, Peter Turcsanyi, Zuzana Mikulkova, Gabriela Gabcova, Renata Urbanova, Petr Gajdos, Veronika Smotkova Kraiczova, Sarka Zehnalova, Tomas Papajik, Eva Kriegova PII: DOI: Reference:

S0145-2126(18)30184-X https://doi.org/10.1016/j.leukres.2018.08.006 LR 6016

To appear in:

Leukemia Research

Received date: Revised date: Accepted date:

31-3-2018 11-7-2018 8-8-2018

Please cite this article as: Manukyan G, Turcsanyi P, Mikulkova Z, Gabcova G, Urbanova R, Gajdos P, Kraiczova VS, Zehnalova S, Papajik T, Kriegova E, Dynamic changes in HLA-DR expression during short-term and long-term ibrutinib treatment in patients with chronic lymphocytic leukemia, Leukemia Research (2018), https://doi.org/10.1016/j.leukres.2018.08.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dynamic changes in HLA-DR expression during short-term and long-term ibrutinib treatment in patients with chronic lymphocytic leukemia

Gayane Manukyan1,4*, Peter Turcsanyi2*, Zuzana Mikulkova1, Gabriela Gabcova1, Renata

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Urbanova2, Petr Gajdos3, Veronika Smotkova Kraiczova1, Sarka Zehnalova3, Tomas Papajik2, Eva Kriegova1# *

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contributed equally

Department of Immunology, Faculty of Medicine and Dentistry, Palacky University

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Olomouc, Czech Republic; 2Department of Hemato-Oncology, Faculty of Medicine and

Department of Computer Science, Faculty of Electrical Engineering and Computer Science,

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Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic;

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VSB-Technical University of Ostrava, Czech Republic; 4Laboratory of Molecular and

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Cellular Immunology, Institute of Molecular Biology NAS RA, Yerevan, Armenia.

Correspondence: Eva Kriegova, Department of Immunology, Faculty of Medicine and

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Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic; e-mail:

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[email protected]

Highlights

Ibrutinib influences the expression of HLA-DR on CLL cells.

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Ibrutinib gradually increases HLA-DR levels on circulating monocyte subsets.

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Immunomodulatory effect of ibrutinib on CLL, T cells, monocytes is time-dependent.

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Abstract There is first evidence about the changes in the kinetics of B cell antigen receptor (BCR) internalisation of neoplastic cells in chronic lymphocytic leukemia (CLL) after the short-term and long-term administration of ibrutinib. We aimed to assess the influence of short-term and

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long-term ibrutinib treatment on the HLA-DR expression on CLL cells, T cells and monocytes. The immunophenotyping of CLL and immune cells in peripheral blood was

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performed on 16 high-risk CLL patients treated with ibrutinib. After early ibrutinib administration, the HLA-DR expression on CLL cells reduced (P=0.032), accompanied by an increase in CLL cell counts in peripheral blood (P=0.001). In vitro culturing of CLL cells

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with ibrutinib also revealed the reduction in the HLA-DR expression at protein and mRNA

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levels (P<0.01). The decrease in HLA-DR on CLL cells after the first month was followed by

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the gradual increase of its expression by the 12th month (P=0.001). A one-month follow-up

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resulted in elevated absolute counts of CD4+ (P=0.002) and CD8+ (P<0.001) T cells as well

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as CD4+ and CD8+ cells bearing HLA-DR (P<0.01). The long-term administration of ibrutinib was associated with the increased numbers of CD4+ bearing HLA-DR (P=0.006) and

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elevation of HLA-DR expression on all monocyte subsets (P<0.004). Our results provide the first evidence of the time-dependent immunomodulatory effect of ibrutinib on CLL and T

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cells and monocytes. The clinical consequences of time-dependent changes in HLA-DR

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expression in ibrutinib treated patients deserve further investigation.

Keywords: chronic lymphocytic leukemia; ibrutinib; HLA-DR; CLL cells; T cells

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1. Introduction Ibrutinib, an irreversible small-molecule inhibitor of both Bruton's tyrosine kinase (BTK) and interleukin-2 inducible kinase (ITK), has shown outstanding efficacy in controlling relapsed/refractory and/or previously untreated CLL [1, 2]. Although the exact mechanisms are not completely understood, there is a growing body of evidence about the

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immunomodulating effects of ibrutinib on different immune cell types and signalling

pathways in various types of hematologic and solid cancers [3, 4, 5]. In CLL, ibrutinib has

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been shown to abrogate CLL cell trafficking, signalling, and adhesion in response to tissue homing chemokines such as CXCL12 and CXCL13 [6, 7, 8]. Moreover, in vitro studies showed the cytotoxic effect of ibrutinib in CLL cells, the inhibition of lymphocyte

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proliferation and DNA replication [6]. It has also been reported that ibrutinib downregulates

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the expression of CD20 antigen in CLL cells, thus limiting the efficiency of combined

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rituximab and ibrutinib treatment [9]. Recent studies have shown that ibrutinib reduces T-cell

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microenvironment in CLL [10, 11].

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pseudo-exhaustion (acquired T-cell dysfunction) as well as disrupts the tumour

Although human leukocyte antigen-DR (HLA-DR) plays a pivotal role in the initiation

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and regulation of an antigen-specific immune response and is expressed constitutively on antigen-presenting cells, such as B cells and monocytes, and activated T cells [12], knowledge

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about the effect of ibrutinib on HLA-DR expression on CLL cells and bystander cells in hematologic malignancies is limited. Moreover, MHC class II molecules may serve as signal-

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transducing receptors on T cells and play a role in T cell anergy induced by a soluble form of antigenic peptide [13]. Regarding neoplastic cells, there is a growing body of evidence that HLA-DR expression on their surface is essential for establishing effective antitumor immunity via CD4+ cell activation [14, 15] and activation of effector cell populations, such as macrophages as shown in the animal model of myeloma [16]. Importantly, a recent study

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showed dynamic changes in BCR internalisation kinetics of leukemic cells after short-term and long-term administration with ibrutinib and acalabrutinib in CLL patients [17]. While the requirement of BCR internalisation for antigen presentation is clear, we studied the effects of the short-term and long-term administration of ibrutinib on the expression of HLA-DR on

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circulating CLL cells as well as bystander T cells and monocytes in patients with CLL.

2. Materials and methods

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2.1 Patients

Peripheral blood samples were collected from 16 patients with CLL (F/M: 5/11; median age 67 yrs) who received ibrutinib treatment and 16 age-matched healthy controls.

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The diagnosis of CLL was established according to the IWCLL (The International Workshop

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Group on CLL) guidelines [18]. For clinical characteristics of enrolled patients see Table 1.

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Fresh blood samples were collected before initiation (<7 days) and after being on ibrutinib

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treatment for one month. In eleven patients with longer follow-up, the blood samples were

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analysed after twelve months of ibrutinib treatment. For in vitro experiments, peripheral blood mononuclear cells (PBMCs) were isolated from the 13 patients with a high percentage of CLL

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cells (median 92%; min-max 66-98%, assessed by CD5+/CD19+ staining), who were not receiving ibrutinib treatment.

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All patients gave their informed consent for the use of peripheral blood, taken

primarily for diagnostic evaluation, for the purpose of this study. The local ethical committee

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approved the study.

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2.2 HLA-DR expression measurement The immunophenotypic analysis by six-color flow cytometry of major immune and malignant cells was performed as reported previously [19]. CLL cells in whole blood were stained with an antibody cocktail, which consisted of anti-HLA-DR-FITC (clone L243), antiCD5-PE (UCHT2), anti-CD20-PE (2H7), and anti-CD19-APC-Cy7 (SJ25C1) (BioLegend). T

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and B cells were stained with anti-CD3-FITC (OKT3), anti-CD4-APC-Cy-7 (RPA-T4), antiCD8-PE-Cy-7 (SK1), anti-CD19-APC-Cy-7 (SJ25C1) monoclonal antibodies. Monocyte

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subsets were stained with anti-CD14-FITC (M5E2) and anti-CD16-PE (B73.1) surface markers. Analysis was performed on a BD FACSCanto II (Becton Dickinson).

Isotype control irrelevant antibodies (MOPC-21) matched FITC, PE, PerCP-Cy5.5,

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PE-Cy7, APC and APC-Cy-7 (BioLegend) were used as negative controls. The main cell

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populations were identified using a sequential gating strategy after the exclusion of doublets

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as follows: CLL cells (CD5+/CD19+), CD4+ T cells (CD3+/CD4+), CD8+ T cells

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(CD3+/CD8+), and monocytes (CD14+). Monocyte subsets were gated as follows: classical

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(CD14+/CD16-), intermediate (CD14+/CD16+), and non-classical (CD14dim/CD16+). Flow cytometry data was analysed using the FlowJo vX0.7 software (Tree Star, Inc, San Carlos,

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CA). The data are presented as a percentage of cells positive for HLA-DR or median

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fluorescence intensities (MFI) of its expression.

2.3 Cell culture

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PBMCs were separated by Ficoll density gradient centrifugation. Before stimulation

with ibrutinib, freshly isolated PBMCs from all studied subjects were starved for 1 hour in a serum-free media. Cells were resuspended at a density of 1×106/mL in 24-well plates in the absence or presence of ibrutinib (10 µM) for 24 hours and 48 hours. After PBMCs harvesting, the cells were analysed using flow cytometry or kept in RNAlater solution for RT-PCR 5

analysis.

2.4 Quantitative RT-PCR RNA extraction from cultured CLL cells and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) was performed for HLA-DRB1 (primers: forward 5´reverse

5´-GCTGCCTGGATAGAAACCAC-3´);

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ACAACTACGGGGTTGTGGAG-3´,

PGK1 was used as a reference gene (forward 5´-CTCAACAACATGGAGATTGG-3´, reverse

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5´-CTTTGGACATTAGGTCTTTGAC-3´) using LightCycler® 480 SYBR Green Master (Roche) were performed as previously described [20].

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2.5 Statistics

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The differences between groups were calculated by Mann-Whitney U-test by R-

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software. The differences between unstimulated and stimulated samples from the same

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of < 0.05 were considered significant.

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subject were calculated using the Wilcoxon non-parametric test for paired samples. P values

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3. Results

3.1 Changes in HLA-DR expression on CLL cells after short-term and long-term treatment

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with ibrutinib

The comparison of HLA-DR expression on CLL cells from patients before ibrutinib

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treatment vs healthy controls revealed no differences in the expression of HLA-DR in the patient´s circulating CLL cells compared to CD19+ B cells from healthy controls (P=0.353), showing high inter-individual variability of HLA-DR expression on CLL cells (Table 2). After one month of treatment, the HLA-DR expression on CLL cells was reduced (P=0.032) (Fig. 1A) accompanied with an increase in CLL cell counts in peripheral blood 6

(P=0.001) (Fig. 1B). The expression of CD20 also reduced (P=0.007) after short-term treatment (Fig. 3). Next, we performed in vitro experiments using PBMCs isolated from a cohort of patients with a high percentage of CLL cells, who were not receiving ibrutinib treatment. After the culturing of CLL cells in the absence or presence of ibrutinib, a markedly reduced HLA-DR expression was observed in the treated samples after 24 hours (P<0.001)

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and 48 hours (P<0.010) (Fig. 2A). These results were confirmed at mRNA level (Fig. 2B).

In the patients with a twelve-month follow-up, HLA-DR (P=0.001) and CD20

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(P=0.004) expression on CLL cells increased after long-term administration (1 yr) compared

to short-term treatment (1 month) (Fig. 3A). The twelve-month follow-up showed a trend in the reduced percentage of CLL cells (P=0.067), which was not associated with the reduction

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in CLL cell absolute counts (P=0.078) (Fig. 3B). CD20 expression at twelve months did not

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and percentage (P=0.004 and P=0.031) (Fig. 3).

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differ from the baseline, however, comparison with the first month it showed increased MFI

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3.2 Changes in HLA-DR expression on T cells after short-term and long-term treatment with ibrutinib

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When the CLL patients and healthy controls were compared, an increased HLA-DR expression on CD4+ cells (P=0.042) and no difference on CD8+ cells (P=0.633) were

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observed in CLL patients (Table 2). Regarding the absolute number of CD4+ and CD8+ cells, a decrease in CD4+ (P=0.027) and no difference in CD8+ (P=0.236) absolute counts in CLL

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patients prior to ibrutinib administration was observed comparing to the healthy controls (Table 2). After one-month of ibrutinib treatment, the absolute counts of CD4+ (P=0.007) and CD8+ (P=0.009) cells bearing HLA-DR were increased, but no differences in HLA-DR expression on CD4+ and CD8+ cells were observed. Nevertheless, markedly elevated absolute 7

counts of CD4+ (P=0.002) and CD8+ (P<0.001) cells were evident in our patients after shortterm treatment with ibrutinib (Fig. 1B). Long-term administration was associated with no differences in HLA-DR expression on CD4+ and CD8+ cells (Fig. 3B). However, increased numbers of CD4+ bearing HLA-DR cells (P=0.006), and unchanged numbers of CD8+ bearing HLA-DR cells were detected after

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1-yr treatment (P=0.695). Absolute counts of CD4+ T cells (P=0.700) were not influenced by long treatment, while CD8+ T cells (P=0.032) were reduced after long-term administration (1

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yr) compared to short-term treatment (1 month) (Fig. 3).

3.3 HLA-DR expression after ibrutinib treatment on monocytes

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Lower HLA-DR expression on classical (CD14+/CD16-) (P=0.020) and intermediate

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(CD14+/CD16+) (P=0.005) monocyte subsets were observed in CLL patients as compared to

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healthy controls; no difference in HLA-DR on non-classical monocytes (P=0.156) as well as

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in absolute counts of all monocyte subgroups was observed (Table 2). After one month of

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ibrutinib treatment, an increased HLA-DR expression on classical (P=0.065), intermediate (P=0.320), non-classical (P=0.173) monocytes were detected. The absolute counts of

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monocytes were not changed after short-term treatment (P=0.289) (Fig. 2B). Long-term administration was associated with a marked increase in HLA-DR

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expression on all monocyte subsets: classical (P<0.001), intermediate (P=0.004), nonclassical (P<0.001) ones (Fig. 3A). The absolute counts of monocytes were not changed after

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long-term treatment (P=0.470) (Fig. 3B).

4. Discussion A recent study reported changes in BCR internalisation kinetics as well as the anergy status of neoplastic cells of CLL patients between short-term and long-term administration of 8

ibrutinib [17]. We addressed the question of whether the expression of HLA-DR, a crucial molecule in antigen presentation, on CLL cells and bystander T cells and monocytes are altered after the ibrutinib treatment in a time-dependent manner. Indeed, a one-month treatment of CLL patients with ibrutinib led to the reduced HLA-DR expression on CLL cells accompanied with an increase in CLL cell counts, which is in line with published data on the

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increase in lymphocytosis after ibrutinib administration [21]. The reduction of HLA-DR on CLL cells was also evident by short-term in vitro culture experiments with ibrutinib.

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There is a growing body of contemporary evidence that CLL cells are efficient antigen-presenting cells [17, 22] able to present complex exogenous antigen or endogenous

CLL cell antigen presented by HLA class II molecules to CD4+ cells [22, 23]. However,

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despite the fact that a portion of CLL cells are actually responsive to external stimuli, a

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substantial segment of CLL patients are characterised by the expansion of clonal B cells that

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have anergic features [24, 25]. Moreover, it has been shown that BCR internalisation of

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ligands that bind to the BCR and their accumulation in endosomes is 3 times more efficient in

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CLL than normal B cells and is highest of all in CLL with anergic BCRs [17]. The authors also showed that short-term treatment with ibrutinib and acalabrutinib resulted in increased

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sIgM levels and a decrease in BCR internalisation efficiency [17]. These data together with our observations on reduced HLA-DR expression on CLL cells lead us to suggest that lower

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BCR internalisation during the first month(s) of ibrutinib administration may contribute to lower antigen processing and peptide loading on MHC class II molecules, and the subsequent

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lower expression of MHC class II to elicit T cell help. This may indeed contribute, together with the deregulation of innate immunity, as a result of targeting BTK [26], to enhanced infection rates in ibrutinib treated patients, occurring predominantly within the first months of administration [2, 27]. Additionally, neoplastic cells with the reduced HLA-DR levels may represent cells with limited proliferative capacity [28]. Nevertheless, it is known that MHC 9

class II signals depend on the differentiation state of these cells [29] and the loss/reduction of MHC class II may also contribute to tumour escape [30]. Regarding the molecular mechanisms of HLA-DR reduction caused by ibrutinib in the setting of CLL, knowledge is limited. The data from class II-restricted antigen presentation by B cells however point to well-controlled intracellular machinery, often regulated by the combined effects of several

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molecular events which follow antigen uptake through the BCR and internalisation of protein antigens [31, 32].

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In our patients with longer follow-up, the decrease in HLA-DR on CLL cells after the

first month was followed by the gradual increase of its expression by the twelfth month. This observation may reflect the recently reported results after long-term administration of

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ibrutinib and acalabrutinib in CLL patients (>1yr), where the authors reported a decrease in

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sIgM levels with an increased BCR internalisation efficiency associated with anergic BCRs

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comparing to short-term administration [17]. The upregulation of HLA-DR antigen during

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long-term treatment of ibrutinib may result from the immune response mediated by IFN-γ and

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other Th1 cytokines [33], since ibrutinib selectively promotes Th1 expansion, thereby potentiating Th1-based immune responses and skewing Th1/Th2 cytokines in CLL [3]. The

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underlying molecular mechanism and clinical consequences of time-dependent changes in HLA-DR expression together with changes in BCR internalisation kinetics in ibrutinib treated

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patients deserve further investigation. Furthermore, there is a growing body of evidence that HLA-DR and CD20 antigens

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are physically and functionally coupled on human B cells [34, 35], and the engagement of these molecules on CLL cells with certain mAbs induced rapid homotypic adhesion and subsequent nonapoptotic cell death [36]. We therefore investigated whether the observed changes in HLA-DR may also be evident for CD20 on CLL cells. Indeed, short-term treatment with ibrutinib causes similar changes in the expression of both markers, HLA-DR 10

and CD20, resulting in a decrease in CD20 expression in most of our patients. In line with our data, recent reports have also shown that short-term administration of ibrutinib downmodulates CD20 expression in vivo [9, 37] and in vitro [38]. During long-term treatment in our patients, the expression of CD20 on CLL cells was gradually increasing similarly as HLA-DR.

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Having observed the changes in HLA-DR expression in CLL cells, we were interested

whether the same effect could be detected in T cells. However, no changes in HLA-DR

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expression on CD4+ and CD8+ T cells during the treatment was observed. Importantly,

increased CD4+ T cell counts were detected during a 12-month follow-up, while an observed increase of CD8+ T cells counts at the treatment initiation were later reduced by long-term

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treatment in the majority of patients. Contrary data was reported on clinical trial cohort

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consisting of predominantly treatment-naive patients with/without TP53 disruption, where a

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decrease of CD4+ and CD8+ T cell counts as well as HLA-DR and CD39 on T-cells were

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reported after 6-month treatment with ibrutinib, thus linking it to the reduction of T cell

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pseudo-exhaustion [10]. In agreement with our results, a recent study showed that ibrutinib markedly increases CD4+ and CD8+ T cell numbers as well as the persistence of activated T

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cells in vivo in CLL patients on clinical trials, which the authors linked to ITK inhibition [39]. Whether the discrepancy between the studies may be linked to heterogeneity of cohorts and/or

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the duration of treatment deserves further investigation. It is likely that the interaction between T and B cells, as mediated by HLA-DR, results in the delivery of activation signals

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to B cells, as reported by others [40]. HLA-DR expression on circulating monocytes in CLL has not yet been investigated.

Multiple roles for BTK in the development and function of myeloid cells have been recently proposed [26], however there are conflicting results on the immunosuppressive properties of ibrutinib on these cells. In a recent study, ibrutinib was unable to antagonise the protecting 11

role of nurse-like cells and the enhanced immunosuppressive phenotype of macrophages, which may mediate ibrutinib resistance in vitro [41]. In another study, ibrutinib disrupted CLL-macrophage interactions by a reduction in macrophage pseudopodia interacting with CLL cells [10]. In our study, marked elevation of HLA-DR on monocytes subsets was observed after long-term treatment with ibrutinib. Regarding the HLA-DR on monocytes,

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there is evidence that its upregulation increases the antigen presentation by these cells thus

contributing to the clearance of pathogens [42]. Whether the recovery of HLA-DR levels on

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circulating monocytes leads to the improvement of cell functioning in the setting of CLL needs to be addressed in future studies.

Our study has certain limitations. The patient cohort is modest thus larger patient

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cohorts are needed to confirm our results. Moreover, no subanalysis on clinical and laboratory

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parameters were performed due to the high heterogeneity and small size of patient subgroups.

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Finally, the molecular mechanisms, functional consequences, and the clinical relevance of

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time-dependent changes of HLA-DR levels on CLL and T cells should be determined in

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Conclusion

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future studies.

Our study provides new insights into the immunomodulatory action of ibrutinib,

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showing dynamic changes in the immune cell composition and its activation, at least during the first year of ibrutinib administration. Further studies are required to determine functional

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consequences of time-dependent changes in HLA-DR expression on CLL cells and bystander immune cells during ibrutinib treatment and the clinical relevance of these findings. Declarations of interest: none Funding: This work was supported by grant of Ministry of Health of Czech Republic (MZ ČR VES16-32339A), in part by MH CZ – DRO (FNOl, 00098892) and LF UP_2018_016. 12

Acknowledgment The authors would like to thank L Kruzova for the performance of routine FISH and

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cytogenetic analysis.

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References [1] R.H. Advani, J.J. Buggy, J.P. Sharman, et al, Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies, J. Clin. Oncol. 31 (2013) 88-94.

chronic lymphocytic leukemia, N. Engl. J. Med. 369 (2013) 32-42.

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[2] J.C. Byrd, R.R. Furman, S.E. Coutre, et al, Targeting BTK with ibrutinib in relapsed

[3] J.A. Dubovsky, K.A. Beckwith, G. Natarajan, et al, Ibrutinib is an irreversible molecular

SC R

inhibitor of ITK driving a Th1-selective pressure in T lymphocytes, Blood. 122 (2013) 2539-2549.

[4] C. Cubillos-Zapata, J. Avendaño-Ortiz, R. Córdoba, et al, Ibrutinib as an antitumor in

patients

with

refractory chronic

U

immunomodulator

leukemia,

N

Oncoimmunology. 5 (2016) e1242544.

lymphocytic

A

[5] J. Molina-Cerrillo, T. Alonso-Gordoa, P. Gajate, et al, Bruton's tyrosine kinase (BTK) as

M

a promising target in solid tumors, Cancer Treat. Rev. 58 (2017) 41-50.

ED

[6] S. Ponader, S.S. Chen, J.J. Buggy, et al, The Bruton tyrosine kinase inhibitor PCI-32765

thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in

PT

vivo, Blood. 119 (2012) 1182-1189.

[7] M.F. de Rooij, A. Kuil, C.R. Geest, et al, The clinically active BTK inhibitor PCI-32765

CC E

targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia, Blood. 119 (2012) 2590-2594.

A

[8] S.S. Chen, B.Y. Chang, S. Chang, et al, BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia, Leukemia. 30 (2016) 833-843. [9] G. Pavlasova, M. Borsky, V. Seda, et al, Ibrutinib inhibits CD20 upregulation on CLL B cells mediated by the CXCR4/SDF-1 axis, Blood. 128 (2016) 1609-1613. 14

[10] C.U. Niemann, S.E. Herman, I. Maric, et al, Disruption of in vivo Chronic Lymphocytic Leukemia Tumor-Microenvironment Interactions by Ibrutinib--Findings from an Investigator-Initiated Phase II Study, Clin. Cancer Res. 22 (2016) 1572-1582. [11] P. Bachireddy, C.J. Wu, Arresting the Inflammatory Drive of Chronic Lymphocytic Leukemia with Ibrutinib, Clin. Cancer Res. 22 (2016) 1547-1549.

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[12] C. Tincat, G.M. Bellistrì, G. Ancona G, et al, Role of in vitro stimulation with lipopolysaccharide on T-cell activation in HIV-infected antiretroviral-treated patients,

SC R

Clin. Dev. Immunol. 2012 (2012) 935425.

[13] H. Kudo, T. Matsuoka, H. Mitsuya, Y. Nishimura, S. Matsushita. Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-

U

dependent kinase inhibitor p27(Kip1). Immunol. Lett. 81(2002):149-155.

N

[14] Y. Xie, A. Akpinarli, C. Maris, et al, Naive tumor-specific CD4+ T cells differentiated in

A

vivo eradicate established melanoma, J Exp Med, 207 (2010) 651-667.

M

[15] J. Yatsuda, A. Irie, K. Harada, et al, Establishment of HLA-DR4 transgenic mice for the

ED

identification of CD4+ T cell epitopes of tumor-associated antigens, PLoS One, 8 (2013) e84908.

PT

[16] A. Corthay, D.K. Skovseth, K.U. Lundin, et al, Primary antitumor immune response mediated by CD4+ T cells, Immunity, 22 (2005) 371-383.

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[17] E.M. Coulter, A. Pepper, S. Mele, et al, In-vitro and in-vivo evidence for uncoupling of BCR internalization and signaling in chronic lymphocytic leukemia, Haematologica, 103

A

(2018) 497-505.

[18] M. Hallek, B.D. Cheson, D. Catovsky, et al, International Workshop on Chronic Lymphocytic Leukemia. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic

15

Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines, Blood, 111 (2008) 5446-5456. [19] G. Manukyan, T. Papajik, P. Gajdos, et al, Neutrophils in chronic lymphocytic leukemia are permanently activated and have functional defects, Oncotarget, 8 (2017) 8488984901.

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[20] T. Tomankova, E. Kriegova, R. Fillerova, et al, Comparison of periprosthetic tissues in

knee and hip joints: differential expression of CCL3 and DC-STAMP in total knee and

SC R

hip arthroplasty and similar cytokine profiles in primary knee and hip osteoarthritis, Osteoarthritis Cartilage, 22 (2014) 1851-1860.

[21] S.E. Herman, C.U. Niemann, M. Farooqui, et al, Ibrutinib-induced lymphocytosis in

U

patients with chronic lymphocytic leukemia: correlative analyses from a phase II study,

N

Leukemia, 28 (2014) 2188-2196.

A

[22] A. Os, S. Bürgler, A.P. Ribes, et al, Chronic lymphocytic leukemia cells are activated and

M

proliferate in response to specific T helper cells, Cell Rep, 4 (2013) 566-577.

ED

[23] R.N. Damle, C. Calissano, N. Chiorazzi, Chronic lymphocytic leukaemia: a disease of activated monoclonal B cells, Best Pract Res Clin Haematol, 23 (2010) 33-45.

PT

[24] C.I. Mockridge, K.N. Potter, I. Wheatley, et al, Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status, Blood, 109

CC E

(2007) 4424-4431.

[25] M. Muzio, B. Apollonio, C. Scielzo, et al, Constitutive activation of distinct BCR-

A

signaling pathways in a subset of CLL patients: a molecular signature of anergy, Blood, 112 (2008) 188-195.

[26] A.N.R. Weber, Z. Bittner, X. Liu, et al, Bruton's Tyrosine Kinase: An Emerging Key Player in Innate Immunity, Front Immunol, 8 (2017) 1454. [27] A.M. Williams, A.M. Baran, P.J. Meacham, et al, Analysis of the risk of infection in 16

patients with chronic lymphocytic leukemia in the era of novel therapies, Leuk Lymphoma, 59 (2018) 625-632. [28] M. Wetzler, B.K. McElwain, C.C. Stewart, et al, HLA-DR antigen-negative acute myeloid leukemia, Leukemia, 17 (2003) 707-715. [29] W. Guo, J.G. Castaigne, N. Mooney, et al, Signaling through HLA-DR induces PKC

IP T

beta-dependent B cell death outside rafts, Eur J Immunol, 33 (2003) 928-938.

[30] K. Töpfer, S. Kempe, N. Müller, et al, Tumor evasion from T cell surveillance, J Biomed

SC R

Biotechnol, 2011 (2011) 918471.

[31] A.M. Avalos, H.L. Ploegh, Early BCR Events and Antigen Capture, Processing, and Loading on MHC Class II on B Cells, Front. Immunol, 5 (2014) 92.

U

[32] L.N. Adler, W. Jiang, K. Bhamidipati, et al, The Other Function: Class II-Restricted

N

Antigen Presentation by B Cells, Front. Immunol, 8 (2017) 319.

A

[33] F.J. Salgado, J. Lojo, C.M. Fernández-Alonso, et al, Interleukin-dependent modulation of

M

HLA-DR expression on CD4and CD8 activated T cells, Immunol. Cell Biol, 80(2002)

ED

138-147.

[34] G. Greicius, L. Westerberg, E.J. Davey, et al, Microvilli structures on B lymphocytes:

PT

inducible functional domains? Int. Immunol, 16 (2004) 353–364. [35] H. Li, L.M. Ayer, M.J. Polyak, et al, The CD20 calcium channel is localized to microvilli

CC E

and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism, J

A

Biol Chem, 279 (2004) 19893-19901.

[36] A. Ivanov, S.A. Beers, C.A. Walshe, et al, Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells, J Clin Invest, 119 (2009) 2143-2159.

17

[37] M. Skarzynski, C.U. Niemann, Y.S. Lee, et al, Interactions between Ibrutinib and AntiCD20 Antibodies: Competing Effects on the Outcome of Combination Therapy, Clin Cancer Res, 22 (2016) 86-95. [38] K. Bojarczuk, M. Siernicka, M. Dwojak, et al, B-cell receptor pathway inhibitors affect

Leukemia, 28 (2014) 1163-1167.

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CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies,

[39] M. Long, K. Beckwith, P. Do, et al, Ibrutinib treatment improves T cell number and

SC R

function in CLL patients, J Clin Invest, 127 (2017) 3052-3064.

[40] H. Tabata, T. Matsuoka, F. Endo, et al, Ligation of HLA-DR molecules on B cells

activation, J Biol Chem, 275 (2000) 34998-35005.

U

induces enhanced expression of IgM heavy chain genes in association with Syk

N

[41] S. Fiorcari, R. Maffei, V. Audrito, et al, Ibrutinib modifies the function of

A

monocyte/macrophage population in chronic lymphocytic leukemia, Oncotarget, 7 (2016)

M

65968-65981.

ED

[42] W. Tillinger, R. Jilch, T. Waldhoer, et al, Monocyte human leukocyte antigen-DR

expression-a tool to distinguish intestinal bacterial infections from inflammatory bowel

A

CC E

PT

disease? Shock. 40(2013) 89-94.

18

Figure legends

Fig. 1. (A) Changes in HLA-DR expression (MFI) on CLL cells, CD4+ and CD8+ T cells, and monocyte subsets (MON), and (B) absolute numbers of CLL cells, CD4+ and CD8+ T cells, CD4+ and CD8+ T cells bearing HLA-DR and total monocytes (MON) in peripheral blood in

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CLL patients after one month on ibrutinib treatment compared to pre-treatment levels. HLADR expression levels on intermediate monocytes were not evaluated in some CLL patients

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N

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due to the low abundance of this subset (<0.2%).

Fig. 2. The percent changes in HLA-DR expression at protein (A) and mRNA (B) levels after

A

24 hrs on culturing with ibrutinib compared to the baseline values.

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Fig. 3. The percent changes in (A) the HLA-DR and CD20 expression (MFI) on circulating

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leukemic cells in CLL patients after one month (light violet columns) and twelve months

N

(dark violet columns) on ibrutinib treatment compared to pre-treatment levels on CLL cells,

A

and HLA-DR on CD4+ T cells, CD8+ T cells, classical MON, intermediate MON, non-

M

classical MON; (B) the absolute counts of circulating cells after one month (light blue

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columns) and twelve months (dark blue columns) on ibrutinib treatment compared to pretreatment levels on CLL cells, CD4+ T cells, CD8+ T cells, CD4+ T cells bearing HLA-DR,

A

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CD8+ T cells bearing HLA-DR, and total MON in CLL patients.

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21

A ED

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N

A

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Table 1 Patient characteristics

N N N N Y Y N Y Y Y Y Y N N Y N

N

U

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unmut unmut unmut unmut unmut unmut unmut unmut unmut mut unmut mut unmut unmut unmut unmut

del(11q) del(17p) TP3 Number Time (Y/N) (Y/N) mutatio of since n treatme diagnosi (Y/N) nt lines s to IBR before (months IBR ) Y N Y 43 2 Y N N 52 2 Y Y Y 52 0 Y N N 139 4 Y Y Y 131 4 N Y N 92 3 N N N 103 2 Y Y N 20 1 N Y Y 63 2 Y N Y 166 6 N Y Y 73 2 N N N 17 1 N Y Y 21 1 N Y N 118 6 N N Y 43 2 Y N N 50 2

A

59 50 59 55 56 64 50 69 50 55 64 77 65 67 60 51

M

M M F M M F F M F M M M F M M M

CK (Y/N)

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P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16

IGVH status

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Patien Gend Age at t er diagnosi s (yrs)

Legend: Y - yes, N - no, unmut – unmutated, mut - mutated, IBR – ibrutinib treatment, CK -

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complex metaphase karyotype

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Table 2 The distribution of (A) HLA-DR expression on T, B cells and subtypes of monocytes (MON), and (B) their absolute cell counts in the peripheral blood of healthy control (C) subjects and CLL patients before the initiation of ibrutinib treatment (pre-treatment levels).

A) Expression (MFI)

Healthy controls (C)

CLL patients

P-value

mean (95%CI)

(C vs CLL)

mean (95%CI)

HLA-DR on CD8+ T cells HLA-DR on B cells#/ CLL cells## HLA-DR on Classical MON

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HLA-DR on Intermediate MON

N

HLA-DR on Non-classical MON

M

CD4+ T cells

Healthy controls (C) mean (95%CI) 1.03 (0.79–1.26) 0.54 (0.31–0.76) 0.11# (0.08–0.14) 0.53 (0.38–0.68) 0.04 (0.02–0.05) 0.05 (0.03–0.08)

A

B) Absolute counts (109 cells/L)

B cells#/ CLL cells##

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Classical MON

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CD8+ T cells

Intermediate MON

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Non-classical MON

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23.7 (19.2–28.3) 37.6 (26.3–49.0) 789## (380–1198) 144 (106–182) 887 (542–1232) 467 (322–612)

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18.3 (16.1–20.4) 30.1 (25.0–35.2) 631# (492–769) 220 (167–273) 1757 (1201–2314) 689 (420–959)

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HLA-DR on CD4+ T cells

CLL patients mean (95%CI) 0.72 (0.43–1.02) 0.72 (0.46–0.99) 23.6## (5.82–41.4) 2.37 (-0.05–4.79) 0.35 (-0.10–0.81) 0.42 (0.004–0.84)

0.042 0.633 0.353 0.020 0.005 0.156

P-value (C vs CLL) 0.027 0.236 0.001 0.765 0.338 0.531