The kinase inhibitors R406 and GS‑9973 impair T cell functions and macrophage‑mediated anti‑tumor activity of rituximab
in chronic lymphocytic leukemia patients

Ana Colado1 · María Belén Almejún1,2 · Enrique Podaza1 · Denise Risnik1 · Carmen Stanganelli3 ·
Esteban Enrique Elías1 · Patricia Dos Santos4 · Irma Slavutsky4 · Horacio Fernández Grecco5 · María Cabrejo5 · Raimundo Fernando Bezares6 · Mirta Giordano1,7 · Romina Gamberale1,7 · Mercedes Borge1,7

Received: 26 June 2016 / Accepted: 18 December 2016
© Springer-Verlag Berlin Heidelberg 2016

Abstract Small molecules targeting kinases involved in B cell receptor signaling are showing encouraging clinical activity in chronic lymphocytic leukemia (CLL) patients. Fostamatinib (R406) and entospletinib (GS-9973) are ATP- competitive inhibitors designed to target spleen tyrosine kinase (Syk) that have shown clinical activity with accept- able toxicity in trials with CLL patients. Preclinical studies with these inhibitors in CLL have focused on their effect in patient-derived leukemic B cells. In this work we show that

Electronic supplementary material The online version of this article (doi:10.1007/s00262-016-1946-y) contains supplementary material, which is available to authorized users.

* Mercedes Borge [email protected]

1 Laboratorio de Inmunología Oncológica, Instituto de Medicina Experimental (IMEX)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)- Academia Nacional de Medicina (ANM), Pacheco de Melo 3081, 1425 Buenos Aires, Argentina
2 Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
3 Servicio de Patología Molecular, Instituto de Investigaciones Hematológicas-ANM, Buenos Aires, Argentina
4 Laboratorio de Genética de Neoplasias Linfoides, IMEX- CONICET-ANM, Buenos Aires, Argentina
5 Departamento de Hematología, Sanatorio Julio Méndez, Buenos Aires, Argentina
6 Sección de Hematología, Hospital General de Agudos Dr. Teodoro Álvarez, Buenos Aires, Argentina
7 Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
clinically relevant doses of R406 and GS-9973 impaired the activation and proliferation of T cells from CLL patients. This effect could not be ascribed to Syk-inhibition given that we show that T cells from CLL patients do not express Syk protein. Interestingly, ζ-chain-associated pro- tein kinase (ZAP)-70 phosphorylation was diminished by both inhibitors upon TCR stimulation on T cells. In addi- tion, we found that both agents reduced macrophage-medi- ated phagocytosis of rituximab-coated CLL cells. Overall, these results suggest that in CLL patients treated with R406 or GS-9973 T cell functions, as well as macrophage-medi- ated anti-tumor activity of rituximab, might be impaired. The potential consequences for CLL-treated patients are discussed.

Keywords Chronic lymphocytic leukemia · GS-9973 · R406 · Syk inhibitors · BCR-associated kinase inhibitors

Abbreviations
BCR B cell receptor
Btk Bruton’s tyrosine kinase CD40L CD40 ligand
CFSE Carboxyfluorescein succinimidyl ester CLL Chronic lymphocytic leukemia
ECL Enhanced chemiluminescence Flt3 Fms-like tyrosine kinase 3
Lck Lymphocyte-specific protein tyrosine kinase M-CSF Macrophage colony-stimulating factor
Rx Rituximab
pAb Polyclonal antibodies PVDF Polyvinylidene difluoride SEM Standard error of the mean Syk Spleen tyrosine kinase
Th T helper
ZAP-70 ζ-chain-associated protein kinase-70

Introduction

Leukemic B cells from CLL patients proliferate in lymphoid tissues in particular areas termed proliferation centers where they are in close contact with stroma, monocyte-derived nurse-like cells and activated T cells [1, 2]. Signals derived from these accessory cells provide a supportive microen- vironment which not only promotes CLL cell survival and proliferation but also protects leukemic cells from cytotoxic therapies favoring the relapse of the disease [3]. T cells seem to play a key role in this microenvironment. Bagnara et al. [4] showed in a murine adoptive transfer model of CLL that the presence of activated autologous T CD4+ cells was necessary for human leukemic cell engraftment, survival and prolifera- tion. Moreover, microscopy analysis of lymph nodes from CLL patients showed that CD3+ cells are present in high numbers and mainly localized within proliferation centers [5– 7], and also that proliferating leukemic cells expressing Ki67 are found preferentially next to activated CD4+ T cells [5, 8]. In line with this, in vitro experiments showed that activated T CD4+ lymphocytes can promote CLL cell proliferation and survival through the secretion of anti-apoptotic cytokines, such as IL-4 [9] or IFN-γ [10], and the expression of the mol- ecule CD40 ligand (CD40L) which interacts with CLL cells through CD40 [6, 8]. Also, CD8+ T cells from CLL patients were shown to inhibit specifically leukemic B cell apoptosis in vitro mediated in part by soluble factors [11].
In the last few years, several small molecules target- ing kinases involved in B cell receptor (BCR) signaling have been developed and tested in clinical trials with CLL patients. R406 is an ATP-competitive kinase inhibitor rela- tively selective to Syk and in a lesser degree to other kinases including Fms-like tyrosine kinase 3 (Flt3) and lymphocyte- specific protein tyrosine kinase (Lck) [12]. In vitro studies showed that R406 can induce CLL cell apoptosis by disrupt- ing BCR signaling and other microenvironmental interac- tions [13, 14]. Fostamatinib disodium (R788; FosD) is the prodrug of R406 available in an oral formulation which was initially developed for the treatment of rheumatoid arthritis [12]. The therapeutic effect of fostamatinib in CLL has been successfully tested in the mouse model of CLL Eμ-TCL1
[15] and in a phase I/II study showing both safety and effi- cacy in patients with B cell non-Hodgkin lymphoma and CLL [16]. More recently entospletinib (GS-9973), another ATP-competitive inhibitor of Syk, with higher selectivity than R406 has been developed [17] and tested in a clinical trial with CLL patients showing clinical activity and good tolerance in relapsed or refractory patients [18]. At the pre- sent, GS-9973 is being tested in clinical trials with patients with different hematological disorders including CLL.
Besides their effects on leukemic cells, kinase inhibitors also affect other cell populations from the immune system. For example, the Bruton’s tyrosine kinase (Btk) inhibitor,
ibrutinib, modulates T cell activation in CLL patients, favoring the differentiation toward the T helper (Th) type 1 profile, while it inhibits Th2 activation [19]. Moreover, we [20] and others [21] have recently described that ibru- tinib impairs macrophage-mediated phagocytosis of leu- kemic cells opsonized with rituximab, which is a central mechanism of the anti-CD20 therapy [22, 23]. R406 was also shown to inhibit graft versus host disease in a mouse model by impairing murine T cell activation directly [24] or by targeting antigen presenting cells [25].
Considering the key role of T cells and macrophages in CLL pathogenesis and therapy, the goal of this work was to study the effect of R406 and GS-9973 on these cells.

Materials and methods

Reagents and antibodies

RPMI 1640 was purchased from Life Technologies (Grand Island, NY, USA), and FCS, penicillin and streptomycin were obtained from GIBCO Laboratories (Grand Island, NY, USA). The human recombinant chemokines CXCL12/ SDF-1, CCL19 and CCL21 were purchased from Pepro- Tech (DF, Mexico). BSA was obtained from Wiener Labo- ratorios (Santa Fé, Argentina).
FITC-, PE- or PerCP-Cy™5.5-conjugated mAbs specific for CD69 (clone FN50), CD40L (clone TRAP-1), CCR7 (clone 3D12) and for CXCR4 (clone 12G5) were purchased from BD Bioscience, Pharmingen (CA, USA). FITC-, PE- or PerCP-Cy5-conjugated mAbs specific for CD3 (clone HIT3a), CD4 (clone OKT4), CD8 (clone HIT8a), CD14 (clone HCD14) and CD25 (clone M-A251) and antibodies with irrel- evant specificity (isotype controls) were obtained from Bio- legend (CA, USA). PE-conjugated mAbs specific for CD56 (clone HLDA6) and CD19 (clone J3-119) were obtained from Beckman Coulter (CA, USA). Annexin-V FITC was obtained from Immunotools (Friesoythe, Germany).
For western blot, the mAb specific for Syk (clone 4D10.1) was obtained from Upstate Biotechnology (NY, USA). Polyclonal antibodies (pAb) specific for phospho- ZAP-70 (Tyr319) and mAb for β-actin (8H10D10) were obtained from Cell Signaling (Danvers, MA, USA). The HRP-conjugated mAb for mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the HRP-conju- gated mAb for rabbit IgG from Jackson ImmunoResearch, Inc (West Grove, PA, USA).
For ELISA assays, human IFN-γ kit was obtained from BD Bioscience, human IL-10 kit from Biolegend and human IL-4 kit was purchased from eBioscience (CA, USA). Carboxyfluorescein succinimidyl ester (CFSE) was purchased from Invitrogen Argentina Ltd (Bs. As. Argen- tine), Monensin (BD GolgiStop™) from BD Biosciences

and DMSO from Sigma-Aldrich. IL-15 and macrophage colony-stimulating factor (M-CSF) were obtained from BioLegend. Rituximab was obtained from Roche Diagnos- tics GmbH (Mannheim, Germany).
Fostamatinib (R406) was provided by Rigel Pharma- ceuticals, and entospletinib (GS-9973) was purchased from Medkoo Biosciences (North Carolina, USA).

CLL patient and healthy donor samples

Peripheral blood samples were obtained from CLL patients and age-matched healthy donors. Informed consent was obtained from all individual participants included in the study in accordance with the Declaration of Helsinki and with Institutional Review Board approval from the National Academy of Medicine, Buenos Aires. CLL was diagnosed according to standard clinical and laboratory criteria. At the time of the analysis, all patients were free from clinically rel- evant infectious complications and were either untreated or had not received treatment for a period of at least 6 months before investigation. Clinical characteristics of CLL patients included in this study are depicted in Supplementary Table 1.

Cell separation procedures and culture

PBMC were isolated from fresh blood samples by cen- trifugation over a Ficoll-Triyosom layer (Lymphoprep, Nycomed Pharma, Oslo, Norway), washed twice with saline and resuspended in complete medium (RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin).
T cells from CLL patients and healthy donors were puri- fied by positive selection using CD3 MicroBeads accord- ing to the manufacturer’s instructions (Miltenyi Biotec, Germany, % purity >97) for western blot analysis of Syk in freshly isolated T cells. Negative selection of T cells was performed for functional assays to avoid CD3 downregula- tion, using a BD FACS Aria II Cell sorter cytometer after incubating peripheral blood cells with mAbs specific for CD19, CD14 and CD56 (% purity >97).

Evaluation of activation markers on TCR‑stimulated T cells

PBMC or purified T cells from CLL patients were pre- treated with DMSO, R406 or GS-9973 (0.1 or 1 µM) for 30 min and then transferred to a 48-well culture plate with immobilized anti-CD3 mAb or isotype control (0.15 µg/ mL). After 24 h of culture, the expression of CD25 and CD69 was evaluated on CD4+ and CD8+ T cells.
CD40L (CD154) expression on CD4+ T cells was meas- ured by adding FITC-conjugated anti-CD154 mAb and monensin (2 µM) to the culture during the stimulation, as
described previously [26]. CD40L expression was analyzed by flow cytometry on CD4+ T cells after 24 h of culture.

Assessment of T cell proliferation

T cell proliferation was evaluated by flow cytometry using the CFSE dilution assay. PBMC or purified T cells from CLL patients were labeled with CFSE 1 µM according to the manufacturer’s instructions. Afterward, cells were pre- treated with DMSO, R406 or GS-9973 (0.1 or 1 µM) for 30 min and then transferred to a 48-well culture plate with immobilized anti-CD3 mAb or isotype control (0.3 µg/mL) or IL-15 (20 ng/ml). After 5 days of culture, cells were col- lected and stained with mAbs specific for CD4 (PerCP- Cy5) and for CD8 (PE). The number of cells that had pro- liferated was determined by gating on the CFSElow CD4+ or CD8+ subset of the viable cells, gated according to FSC and SSC parameters criteria.

Chemotaxis assay and chemokine receptor evaluation

×
PBMC were resuspended in RPMI 1640 supplemented with 1% FCS and treated with DMSO, R406 or GS-9973 (1 or 5 µM) for 30 min. Then, cells were used to evaluate CCR7 and CXCR4 by flow cytometry and to evaluate chemotaxis response toward CCL19, CCL21 and CXCL12. Chemot- axis assay was performed by using the Transwell System in 96-well Transwell plate with pore size 5 µm and polycarbon- ate membranes (Costar, Corning Incorporated, NY, USA). 70 µL of medium containing 0.5 106 cells was placed in the upper chamber, and 200 µL of medium alone (control) or medium with CCL19, CCL21 or CXCL12 was placed in the lower chamber. Each condition was performed in dupli- cate. After 2 h of culture at 37 °C, migrating cells in the lower chamber were counted by flow cytometry as the number of cells acquired in 30 s under a defined flow rate. Migration index was calculated by determining the ratio of migrated T cells (CD3+ CD4+ or CD3+ CD8+) in response to the chemokine versus spontaneous migrated T cells (control well with medium alone), taking the spontaneous migration in control wells as 100%. The spontaneous migration in con- trol wells was always close to 1% of CD3+ cells placed in the upper compartment. CXCL12, CCL19 and CCL21 concen- tration used for the assays was 1 µg/mL [27].

Western blot

×
×
Whole-cell lysates were obtained from 3 106 purified T or B cells from CLL patients and healthy donors using 60 µL of loading buffer 1 5% β-mercaptoethanol. Lysates were boiled at 99 °C for 5 min, and 30 µL of the protein extracts were separated on a standard 10% SDS-PAGE and trans- ferred to polyvinylidene difluoride (PVDF) membranes.

Fig. 1 R406 and GS-9973 impair the expression of CD25, CD69 and CD40L on CD3-stimulated T cells from CLL patients. PBMC from CLL patients were pretreated with R406, GS-9973 or DMSO (vehi- cle) for 30 min and then transferred to a well with immobilized anti- CD3 mAb (0.15 µg/mL) or the corresponding isotype control anti- body. After 24 h of culture, CD25, CD69 and CD40L expression on T cells was analyzed by flow cytometry. a–c The figures show the val-
ues of the MFI of CD25, CD69 and CD40L on CD4+ T cells. Repre- sentative dot plots are shown. d–e The figures show the values of the MFI of CD25 and CD69 on CD8+ T cells. White circles correspond to DMSO-treated cells, black circles to R406-treated cells and gray circles to GS-9973-treated cells. **p < 0.01, ***p < 0.001, Friedman test followed by Dunn’s multiple comparison post test

± =
Fig. 2 R406 and GS-9973 impair INF-γ, IL-4 and IL-10 pro- duction by T cells from CLL patients. Purified T cells from CLL patients were pretreated with R406, GS-9973 or DMSO (vehicle) for 30 min and then transferred to a well with immobilized anti-CD3 mAb (0.15 µg/mL) or the corresponding isotype control antibody.
After 24 h of culture, INF-γ (a), IL-4 (b) and IL-10 (c) were meas- ured in culture supernatants by ELISA. Results are shown as the mean standard error of the mean (SEM), n 8. *p < 0.05, signifi- cance was determined using Friedman test followed by Dunn’s multi- ple comparison post test

Membranes were then blotted with antibodies against Syk followed by HRP-conjugated anti-mouse IgG. Specific bands were developed by enhanced chemiluminescence (ECL) method. The same membrane was blotted with mAb anti-β-actin followed by HRP-conjugated anti-mouse IgG to compare the total amount of protein in each sample.
For phospho-ZAP-70 evaluation, T cells from CLL patients and age-matched healthy donors were purified by negative selection and then pretreated with DMSO, R406 or GS-9973 (1 µM) for 30 min in RPMI 1640 1% FCS. Then, cells were stimulated with mouse mAb anti-CD3 (6 µg/mL)/anti-CD28 (1 µg/mL) and anti-IgG mouse pAb (15 µg/mL) or the anti-IgG mouse alone (control). After 5 min of stimulation, cells were washed with cold PBS, then protein extracts were obtained as described before, separated on a standard 12% SDS-PAGE and transferred to PVDF membrane. Membranes were then blotted with antibodies against phospho-ZAP-70 and β-actin, followed by HRP-conjugated anti-rabbit IgG or anti-mouse IgG, respec- tively. Specific bands were developed by ECL.

Phagocytosis assay

Macrophages were differentiated from healthy donors’ or chronic lymphocytic leukemia patients’ monocytes by cultur- ing them for 5 days in RPMI 1640 with 10% FCS and M-CSF (50 ng/mL). For the phagocytosis assay, macrophages were treated for 30 min with R406, GS-9973 or DMSO and then CLL cells, that were previously labeled with CFSE (1 μM) and coated or not with rituximab (50 μg/mL), were added to the culture. After 2 h, macrophages were trypsinized and the phagocytosis was evaluated by flow cytometry. Macrophages were determined by morphology in the FSC-H and SSC-H dot plot. The expression of CD20 was evaluated in CLL cells by flow cytometry after 48 h of treatment with DMSO, R406 or GS-9973. Phagocytosis was also evaluated by confocal
microscopy. To this aim, after the phagocytosis assay, mac- rophages were trypsinized, stained with anti-CD14-PE mAb and then centrifuged onto cytospin slides and coverslips were mounted using Fluoromount-G (Sigma). Immunofluores- cence images were acquired with a FluoView FV1000 con- focal microscope (Olympus, Tokio, Japan) using a Plapon
×
60 1.42 NA oil immersion objective, and images were ana- lyzed using the Olympus FV10-ASW software.

Statistical analysis

Statistical significance was determined using the nonpara- metric tests: Friedman test followed by the Dunn’s post test. In all cases, p < 0.05 was considered statistically sig- nificant. Data were analyzed using the GraphPad Prism software version 6.01.

Results

R406 and GS‑9973 impair the expression of activation markers and cytokine‑secretion in response to TCR/ CD3 stimulation on T cells from CLL patients

To evaluate if the kinase inhibitors R406 and GS-9973 affect the activation of T cells, PBMC from CLL patients were cultured with immobilized anti-CD3 mAb in the pres- ence of each inhibitor or vehicle (DMSO). The expression of the activation markers CD25, CD69 and CD40L was evaluated on T cell populations at 24 h by flow cytometry. Results from Fig. 1 show that the up-regulation of these markers induced by polyclonal activation of T cells was impaired by R406 or GS-9973 at 1 µM, which is a clini- cally relevant concentration for both drugs [16, 18], both in CD4+ (Fig. 1a–c) and CD8+ T cells (Fig. 1d–e).

Fig. 3 R406 and GS-9973 impair the proliferation of T cells from CLL patients in response to TCR or IL-15 stimulation. PBMC from CLL patients were labeled with CFSE and then pretreated with R406, GS-9973 or DMSO (vehicle) for 30 min. Then, cells were transferred to a well with immobilized anti-CD3 mAb (0.3 µg/mL) or to a well containing IL-15 (20 ng/mL). After 5 days of culture, cells were col- lected, stained with specific mAb for CD4 (PeCy-5) or CD8 (PE) and then analyzed by flow cytometry. a–b The figures show the percent- age of CFSElow CD4+ T cells from the total CD4+ viable lympho-
cytes, stimulated with anti-CD3 (a) or with IL-15 (b), histograms from representative experiments are shown. c–d The figures show the percentage of CFSElow CD8+ T cells from the total CD8+ viable lym- phocytes, stimulated with anti-CD3 (c) or with IL-15 (d). White cir- cles correspond to DMSO-treated cells, black circles to R406-treated cells and gray circles to GS-9973 treated cells. *p < 0.05, **p < 0.01,
***p < 0.001, Friedman test followed by Dunn’s multiple comparison post test

Similar results were obtained when purified T cells instead of PBMC were used (Supplementary Fig. 1). Importantly, this inhibition was not due to a decrease in cell viability as shown in Supplementary Fig. 2.
The secretion of cytokines (IFN-γ, IL-4 and IL-10) by anti-CD3-activated T cells from CLL patients was also impaired by R406 and GS-9973 (Fig. 2).
R406 and GS‑9973 impair T cell proliferation in response to TCR/CD3 and IL‑15 stimulation

To determine if R406 and GS-9973 were also able to inhibit T cell proliferation, PBMC from CLL patients were labeled with CSFE before activation on immobilized anti-CD3 as described above. As shown in Fig. 3a, c, both inhibitors

Fig. 4 Effects of R406 and GS-9973 on T cell migration and on the expression of chemokine receptors. PBMC from CLL patients were pretreated with R406, GS-9973 or DMSO (vehicle) for 30 min. After, the migratory response toward CCL19 (a), CCL21 (b), CXCL12 (c) (1 µg/mL in RPMI 1640 1% FCS) was evaluated using the Transwell system assay. The figures show the percentage of T cell migration,
calculated as the number of T cells that migrated in response to the chemokine over the number of T cells that migrated spontaneously. The expression of CCR7 (d) and CXCR4 (e) in T cells was evaluated by flow cytometry. Representative histograms are shown. Results are shown as the mean SEM, n 10. *p < 0.05, Friedman test fol- lowed by Dunn’s multiple comparison post test

± =
impaired CD4+ and CD8+ T cell proliferation after 5 days in culture, without significant induction of apoptosis (Sup- plementary Fig. 3). Similar results were obtained when purified T cells instead of PBMC were used (Supplemen- tary Fig. 4). Moreover, inhibition of both activation and proliferation was observed in CD3-stimulated T cells from age-matched healthy donors (Supplementary Fig. 5).
Also, R406 and GS-9973 inhibited T cell prolifera- tion induced by IL-15 (Fig. 3b, d), a cytokine involved in the homeostatic proliferation of memory T cells [28, 29],
indicating that the effect of these inhibitors on T lympho- cytes is not restricted to TCR signaling.

High doses of R406 and GS‑9973 impair T cell migration in response to CCL21, CCL19 and CXCL12

Then we asked if R406 and GS-9973 could modify T cell response to chemokines that regulate their homing to lymphoid organs. To this aim, Transwell chemotaxis assays were per- formed as previously described [27] in the presence of different

a pT cells
pB
cells
pCLL
cells

H.D.
CLL #7
CLL #8
CLL #9
CLL #10
H.D.
CLL #9
CLL #10

72 KDa Syk

42 KDa β -actin

b pT cells pCLL cells

CLL CLL
#1 #2
CLL CLL
#3 #4
CLL CLL #5 #6
CLL CLL CLL CLL #3 #4 #5 #6

72 KDa Syk

42 KDa β -actin

c
pT cells
CLL#1
pT cells
CLL#2
pT cells
H.D.

pCLL cells

- + - + - +
- anti-CD3
72 KDa Syk

42 KDa β -actin

d

pZAP-70
(70KDa)
β -actin
(42 KDa)

pZAP-70
(70KDa)
β-actin
(42 KDa)

T cells from CLL patient # 17

T cells from CLL patient # 14

anti-CD3
R406 (1µM) - +
- - +
+ +
- anti-CD3
R406 (1µM) - +
- - +
+ +
-
GS-9973 (1µM) - - - + GS-9973 (1µM) - - - +

± =
◂Fig. 5 CLL T cells do not express Syk by western blot, and ZAP- 70 phosphorylation is diminished by R406 and GS-9973. a–b T cells from CLL patients and age-matched healthy donors were purified by positive selection using MACS, Miltenyi, MicroBeads kit (>97%). Purified T lymphocytes were used to obtain protein extracts, and subsequently the expression of Syk was evaluated by western blot. c T cells were purified by negative selection using a BD FACS Aria II Cell sorter cytometer (>97%) from PBMC from CLL patients and age-matched healthy donors and then cultured with plate-bound anti- CD3 mAb (0.3 µg/mL). After 5 days of culture, Syk expression was evaluated by western blot. Purified CLL cells were used as positive control and β-actin as charge control. d T cells from CLL patients were purified by negative selection using a BD FACS Aria II Cell sorter cytometer (>97%). Then, T cells were pretreated for 30 min with DMSO, R406 or GS-9973 (1 µM) and then stimulated for 5 min with soluble anti-CD3/anti-CD28 mAbs. Then, phospho-ZAP-70 expression was evaluated by western blot. Specific bands were devel- oped by ECL. Western blots results from patients #17 and 14 are shown. Bands on the immunoblots were quantified using the ImageJ software (NIH Image). H.D.: healthy donors. Results are shown as the mean SEM of the ratio pZAP-70/β-actin, n 5. *p < 0.05, Friedman test followed by Dunn’s multiple comparison post test

concentrations of R406, GS-9973 or the vehicle of the drugs. The chemotactic response of T cells (CD3+), expressed as T cell migration index, is depicted in Fig. 4. The concentrations of the chemokines used in this experiment were the optimal concentrations for the Transwell migration assay for T cells determined in previous studies [27]. We found that migra- tion toward CCL19, CCL21 and CXCL12 was significantly reduced by 5 µM of R406, while GS-9973 5 µM only reduced the migration toward CCL19 and CCL21 (Fig. 4a–c). The inhibition was similar both in CD4+ and CD8+ T cells (not shown). This effect was not due to a decrease in the expression of the chemokine receptors CCR7 and CXCR4 (Fig. 4d, e).

Syk is not expressed in T cells from CLL patients, and ZAP‑70 phosphorylation is impaired by R406 and GS‑9973 after TCR stimulation

Mature T cells from peripheral blood do not normally express Syk [30–32]. Nevertheless, Syk is expressed in T cells during ontogeny in the thymus [30] and also in mature T cells from systemic lupus erythematosus patients [33], T cell lympho- mas [31] and patients with deficiencies in ZAP-70 [32, 34]. In those patients, Syk actively participates in the signal transduc- tion through the TCR [33]. Given that R406 and GS-9973 are mainly selective for Syk, we evaluated the expression of this kinase in purified T cells from CLL patients by western blot. As shown in Fig. 5a we found, as previously described [35], high levels of Syk in B cells from CLL patients. On the con- trary, Syk expression was not detected in either freshly isolated (Fig. 5a, b) or anti-CD3-activated (Fig. 5c) T cells from CLL patients. These results indicate that the effect of both R406 and GS-9973 inhibitors on T cell activation, proliferation and migration is due to the inhibition of kinase/s other than Syk. A previous report showed that concentrations of R406 between
⦁ and 2 µM were sufficient to inhibit ZAP-70 phosphoryla- tion, a kinase involved in TCR signaling [12]. So, we evalu- ated the effect of R406 and GS-9973 on ZAP-70 phospho- rylation after TCR stimulation in purified T cells from CLL patients and age-matched healthy donors. We found that both R406 and GS-9973 at 1 µM impaired ZAP-70 phosphoryla- tion in TCR-stimulated T cells from CLL patients and healthy donors (Fig. 5d and Supplementary Fig. 6).

R406 and GS‑9973 impair macrophage‑mediated phagocytosis of rituximab‑coated CLL cells

=
Anti-CD20 mAb are part of the current standard therapy for CLL patients [36]. The chemoimmunotherapy includ- ing fludarabine (a purine analog), cyclophosphamide (an alkylating agent) and rituximab (an anti-CD20 mAb) is the standard first-line treatment for patients with good physical condition. A combination with an anti-CD20 mAb together with chlorambucil (an alkylating agent) is usually applied to non-fit patients given its lower toxicity [36]. Thus, consider- ing that Syk participates in the signaling through Fcγ recep- tors in myeloid cells [37], we tested the ability of R406 and GS-9973 to impair macrophage-mediated phagocytosis of rituximab-coated CLL cells, which is known to be a central mechanism in the anti-tumor activity of anti-CD20 antibodies [22, 23]. To this aim, human macrophages were differentiated from healthy donors’ monocytes by incubation with M-CSF (50 ng/mL) for 5 days. Then, the phagocytosis assay of CFSE- labeled rituximab-coated CLL cells was performed as previ- ously described [20] in the presence or absence of R406 or GS-9973. Uptake of CFSE-CLL cells was evaluated by flow cytometry and confocal microscopy. As shown in Fig. 6a, b, both R406 and GS-9973, at clinically relevant concentrations, impaired macrophage phagocytosis of rituximab-coated CLL cells. This inhibitory effect was also observed on macrophages differentiated from CLL samples (n 8, p < 0.05 for 1 and
⦁ µM for R406 and GS-9973, respectively, Friedman test
followed by Dunn’s multiple comparison post test). Dot plots from a representative experiment are shown in Fig. 6c.
In addition, we found that R406 induced a slight down- regulation of CD20 expression on CLL cells, confirming a previous report [38], while GS-9973 had no effect (Supple- mentary Fig. 7).

Discussion

In the last few years, small-molecule inhibitors of kinases involved in BCR signaling have been developed and tested for the treatment of patients with B cell malignancies [39]. Among them, the Btk inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib were approved for the treatment of CLL patients in 2014, the first as monotherapy and the second

=
Fig. 6 R406 and GS-9973 impair rituximab (Rx)-coated CLL cells phagocytosis by human macrophages. a Human macrophages were obtained by culturing monocytes from healthy donor’s peripheral blood for 5 days in RPMI 1640 supplemented with 10% FCS and M-CSF (50 ng/mL) in a 48 well-plate. At day five, human mac- rophages were treated for 30 min with R406, GS-9973 or DMSO (vehicle) and then CLL cells, that were previously labeled with CFSE (1 µM) and coated or not with Rx (50 µg/mL), were added to the culture. After 2 h, macrophages were trypsinized and evaluated by flow cytometry and phagocytosis was calculated as the percent-
age of CFSE+ macrophages. Results are shown as the mean SEM, n 10. *p < 0.05, **p < 0.01, ***p < 0.001, Friedman test fol- lowed by Dunn’s multiple comparison post test. b Macrophages were stained with anti-CD14-PE mAb, and preparations were analyzed by confocal microscopy. 3 magnification from the original images is shown in the insert. Representative images of the experiment are shown. c The same phagocytosis experiment was performed using macrophages differentiated from CLL patients’ monocytes. Repre- sentative dot plots are shown

±
×
in combination with rituximab, and have generated great expectation for these and other kinase inhibitors due to the manageable toxicity profile and encouraging clinical effec- tiveness observed with these agents [39].
Although most of preclinical studies of BCR-associated kinases inhibitors in CLL have focused on their effects on patient’s malignant B cells, it is now becoming evident that these inhibitors also affect other immune cells in CLL

patients. Thus, ibrutinib affects functions on T cells [19], NK cells [40] and macrophages [20, 21] by targeting other kinase/s different from Btk. Also, idelalisib alters cytokine production by T and NK cells [41] and phagocytosis by myeloid cells [21].
In this study we found that the kinase inhibitors R406 and GS-9973 significantly impair the activation and prolifera- tion of T cells from CLL patients in response to TCR stimu- lation or IL-15. Likewise, both inhibitors markedly decrease the expression of CD40L and the secretion of IL-4 and IFN- γ, key molecules for CLL-B cell survival and growth. Given the relevance of activated T cells to support CLL progres- sion, our results suggest that R406 and GS-9973 may be successful in the treatment of CLL patients because of their effect not only on the malignant B cell clone but also on T cells from the supportive microenvironment.
Although R406 and GS-9973 have been developed as Syk inhibitors, their effects on T cells described in the pre- sent report could not be ascribed to Syk. Indeed, we found that neither freshly isolated nor activated T cells from CLL patients express Syk. Although we do not know the specific target/s of these inhibitors on T cells, it has been reported that concentrations of R406 between 0.4 and 2 µM are suf- ficient to inhibit other kinases such as Lck, which is respon- sible for ZAP-70 phosphorylation on T cells in response to TCR stimulation [12]. GS-9973, although it was described to be a more selective inhibitor for Syk than R406, can also inhibit Lck and ZAP-70 at higher concentrations [17]. In this work we found that ZAP-70 phosphorylation was impaired by 1 µM of R406 and GS-9973 on T cells from CLL patients and healthy donors upon TCR stimulation, suggesting that Lck activity might be impaired at that con- centration of the drugs. Importantly, patients treated with these agents achieve a peak plasma concentration of 1.6 µM for R406 [16] and 3.6 µM for GS-9973 [18], so it is possible that multiple kinases might be affected during therapy.
The strong impairment in T cell functions induced by
R406 and GS-9973 described here might be particularly detrimental in CLL patients given their known susceptibil- ity to infections and the requirement for long-term therapy with kinase inhibitors [42]. At the moment, there is no clear evidence showing that these drugs enhance the susceptibil- ity to infections in CLL patients. However, it is possible that long-term effects on T cell functions are not evident in the published trials’ results with R406 and GS-9973 in CLL patients given their short time of follow-up [16, 18]. Nevertheless, during a Phase II study of R406 in rheuma- toid arthritis patients, an increased incidence of upper res- piratory tract infections, not associated with neutropenia, was reported [43]. Remarkably, a Phase II trial combining GS-9973 and idelalisib was halted due to a high incidence of pneumonitis [44]. The immune defect responsible for this observation was not determined yet. And, although
pneumonitis was already described in patients treated with idelalisib and inhibition of regulatory T cells by this drug seems to be involved [45], it is possible that an immune defect caused by the decrease in T cell function due to GS-9973 could also be responsible for the high incidence of pneumonitis observed in the combinatory treatment. Results from the ongoing trials with GS-9973 in CLL patients with longer periods of follow-up will probably help to clarify this point.
Finally, it is well known that Syk participates in Fc receptor signaling as demonstrated by the fact that murine macrophages deficient for Syk are unable to phagocyte IgG-coated particles [46]. Accordingly, we observed that R406 and GS-9973 impaired macrophage phagocytosis of rituximab-coated CLL cells, suggesting that Syk inhibitors should not be administrated concomitant with mAb target- ing CLL cells to preserve their efficacy.
Overall, in this work we have shown that R406 and GS-9973 have strong effects on T cell and macrophage functions and that this must be considered in long-term therapies with these drugs and in combinatory treatment with mAbs.

Acknowledgements This work was supported by grants from Fondo para la Investigación Científica y Tecnológica (PICT 1074/2013 and PICT 1310/2012), Ministry of Science, Technology and Innovation, Argentina. We are indebted to Beatriz Loria, María Tejeda and Fed- erico Fuentes for their technical assistance.

Compliance with ethical standards

Conflict of interest Romina Gamberale had received compensation as speaker for Janssen and Bristol-Myers Squibb, as speaker and con- sultant for Roche, and as a member of the advisory board of AbbVie. Raimundo Fernando Bezares had received compensation as speaker from Novartis, Varifarma and Roche. All the other authors declare that they have no conflict of interest.

Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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