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DISSECTION OF THE EFFECTS OF JAK AND BTK INHIBITORS ON THE FUNCTIONALITY OF HEALTHY AND MALIGNANT LYMPHOCYTESTom Hofland1,2*, Iris de Weerdt1,2*, Hanneke ter Burg1,2, Renate de Boer1,2, Stacey

Tannheimer3, Sanne H. Tonino2,4, Arnon P. Kater2,4, Eric Eldering1,4

1Department of Experimental Immunology, Amsterdam UMC, University of Amsterdam, Amsterdam2Department of Hematology, Amsterdam UMC, University of Amsterdam, Amsterdam3Gilead Sciences, Foster City, CA4Lymphoma and Myeloma Center Amsterdam, LYMMCARE, Amsterdam

*These authors contributed equally

J Immunol 2019;203:2100-2109

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ABSTRACTDespite the emergence of small molecule inhibitors, current treatment strategies for chronic

lymphocytic leukemia (CLL) are not curative, and the search for new therapeutic modalities

continues. Pro-survival signaling derived from the micro-environment is often mediated via

JAK signaling. However, whether JAK inhibitors are useful in CLL therapy has not been studied

extensively. JAK inhibitors are valuable therapeutic agents in myelofibrosis and show promising

results in graft-versus-host-disease. However, JAK inhibition is associated with an increased

infection risk, presumably due to the effect on other immune cells, a feature shared with other

kinase inhibitors used for CLL treatment, like the BTK inhibitor ibrutinib and PI3Kδ inhibitor

idelalisib.

We compared functional effects of the JAK1/2 inhibitors momelotinib and ruxolitinib, BTK

inhibitors ibrutinib and tirabrutinib and PI3Kδ inhibitor idelalisib on malignant CLL cells, but

also healthy human T, B and NK lymphocytes. We found several interesting differences among

the inhibitors, apart from expected and well–known effects. Momelotinib, but not ruxolitinib,

blocked cytokine-induced proliferation of CLL cells. Momelotinib also reduced B cell receptor

(BCR) signaling in contrast to ruxolitinib, indicating that these JAK inhibitors in fact have a distinct

target spectrum. In contrast to tirabrutinib, ibrutinib had inhibitory effects on T cell activation,

probably due to ITK inhibition. Remarkably, both BTK inhibitors stimulated IFN-γ production in a

mixed lymphocyte reaction. Collectively, our results demonstrate that kinase inhibitors directed at

identical targets may have differential effects on lymphocyte function. Their unique profile could

be strategically employed to balance desired versus unwanted lymphocyte inhibition.


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DIFFERENTIAL EFFECTS OF KINASE INHIBITORS ON LYMPHOCYTES

INTRODUCTIONChronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant B cells

in blood, lymph nodes, and bone marrow1. Despite the development of targeted compounds

and immunotherapies to eradicate CLL cells, none of the current treatments for CLL is curative.

The Bcl-2 inhibitor venetoclax and Bruton tyrosine kinase (BTK) inhibitor ibrutinib have shown

substantial clinical efficacy in most CLL patients2,3. However, mutations leading to drug resistance

have already been described for both drugs, and because of the high economic burden of lifelong

treatments, alternative strategies leading to actual curative treatments should still be pursued4-7.

Most treatments in CLL are counteracted by the tumor-supportive microenvironment in

secondary lymphoid organs, where CLL cells receive prosurvival signaling from surrounding

stromal cells, T cells and macrophages1,8-10. Therefore, disrupting prosurvival signaling within the

microenvironment is a sensible therapeutic aim in CLL treatment. T cell-derived IL-4 and IL-21

are known survival factors that both signal via JAK11,12. Inhibitors of JAKs have been developed,

most notably momelotinib and ruxolitinib (both targeting JAK1 and JAK2). Ruxolitinib has been

approved by the Food and Drugs Administration and European Medicine Agency for the treatment

of myelofibrosis. Therapeutic inhibition of JAKs with these inhibitors has shown significant

clinical response in myelofibrosis patients, leading to reduced spleen sizes and improved overall

survival13-16. More recently, JAK inhibitors have also shown promising clinical results in graft-versus-

host disease (GvHD), leading to a reduction of steroid need17,18. In CLL, the biological activity of

JAK inhibitors is demonstrated by lymphocyte redistribution out of the lymph nodes and disease

stabilization. Although JAK inhibitors lack efficacy as monotherapeutic agents, based on the pro-

survival contribution of IL-4 and IL-21, combination strategies involving JAK inhibitors may improve

clinical responses in CLL treatment19-21.

As JAK signaling plays a central role in the function of many immune cells, it is not surprising that

JAK inhibitors have been shown to modulate the function of T cells, NK cells and dendritic cells22-

25. In addition, other kinase inhibitors currently used or studied for CLL treatment also display

side effects by the inhibition of off-target kinases. For example, ibrutinib has well-documented

off-target effects on T cells by binding to IL-2-inducible-tyrosine kinase (ITK)26,27. Idelalisib, a

PI3Kδ inhibitor used to treat refractory CLL patients, alters T cell function through modulation of

TCR signaling, possibly explaining the increased amount of atypical infections and autoimmune

complications observed in patients treated with idelalisib28-30. To assess the clinical potential of a

kinase inhibitor for CLL therapy, the combined on- and off-target effects on both malignant and

healthy cells need to be taken into account. In this study, we perform comparative studies of the

effects of JAK, BTK and PI3Kδ inhibitors on CLL cells and healthy immune cells. We determine

both beneficial and detrimental effects of all kinase inhibitors, and explore whether therapeutic

rationales exist to use combinations of these inhibitors not only for CLL therapy, but also for

other diseases.

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MATERIALS AND METHODSPatient and healthy donor samplesPeripheral blood samples from untreated CLL patients were collected at the Amsterdam

University Medical Centers (Amsterdam UMC), location AMC in Amsterdam, the Netherlands.

Healthy donor PBMC were isolated from buffy coats obtained from Sanquin Blood Supply,

Amsterdam, the Netherlands. Ethical approval was provided by the medical ethical committee

at the Amsterdam UMC, location AMC and written informed consent was obtained in accordance

with the Declaration of Helsinki. PBMC from CLL patients and healthy donors were isolated and

cryopreserved as described earlier31.

Cell linesNIH-3T3 fibroblasts (ACC #number 59, Deutsche Sammlung von Mikroorganismen und

Zellkulturen, Braunschweig, Germany) and 3T40L (NIH-3T3 fibroblasts expressing CD40L) were

cultured IMDM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FCS and 1%

penicillin/streptomycin.

CompoundsThe JAK inhibitors momelotinib and ruxolitinib, the BTK inhibitor tirabrutinib and the PI3Kδ inhibitor

idelalisib were all obtained from Gilead Sciences (Foster City, CA). The BTK inhibitor ibrutinib was

purchased from Selleckchem (Houston, TX). Venetoclax (ABT-199) was purchased from Sanbio

B.V. (Uden, the Netherlands). Fludarabine was purchased from Sigma-Aldrich (St Louis, MO). All

compounds were used in concentrations close to their EC50 values or clinically relevant levels.

Proliferation and chemoresistance of CLL cellsFor proliferation experiments, PBMC from CLL patients were labeled with 0.5mM CFSE (Thermo

Fisher Scientific), and cultured for 5 days on either NIH-3T3 or 3T40L cells with or without 25 ng/

ml rIL-21 (Invitrogen, Carlsbad, CA). Cultures were measured on a FACS Canto (BD Biosciences, San

Jose, CA, USA) and data were analyzed using FlowJo Version 10. To study chemoresistance of CLL

cells, PBMC from CLL patients were cocultured with NIH-3T3 or 3T40L cells for 3 days. Cells were

treated with venetoclax or fludarabine for 24 hours. Target cell death was analyzed by incubating

cultures with DiOC6 (Thermo Fisher Scientific) and propidium iodide (PI) (Sigma-Aldrich). Cells were

analyzed on a FACSCanto flow cytometer. Data was analyzed using FlowJo Version 10.

CLL cell viability and Western blot analysisCLL-derived PBMC were preincubated for 1 hour with inhibitors, and stimulated for 24 hours

with IL-4 (10 ng/ml, Bio-Techne, Minneapolis, MN) and during the last 30 minutes with anti-IgM

(20 µg/ml; BioLegend, San Diego, CA). Viability was measured by FACS using DiOC6/PI staining as

described above. For Western blot, cell lysates were prepared by lysing in RIPA buffer (150 mM

NaCl, 1 mM EDTA, 50 mM Tris-HCl [pH 7.4], 0.1% SDS, and 1% NP-40) and subsequently subjected

to 10 seconds of sonication in a Branson sonicator (Danbury, CT). Lysates were analyzed by SDS–


81


PAGE. Western blot was performed using the following abs: rabbit anti-p-STAT6, rabbit anti-

pErk, rabbit anti-pAkt (Ser473), rabbit anti-pS6, mouse anti-S6 (all from Cell Signaling Technology,

Danvers, MA) and goat anti-actin (Santa Cruz Biotechnology, Dallas, TX).

Proliferation, IgM/IgG production and differentiation of healthy B cellsHealthy donor PBMC were stained with CFSE, and stimulated with CpG ODN2006 (1 mg/ml,

Invitrogen) and IL-2 (100 U/ml) for 6 days. Fresh drugs were added after 3 days of stimulation.

Cells were stained with the following abs for flow cytometry: CD19-PerCP-Cy5.5, CD20-

allophycocyanin-H7, IgD-PE, CD27-allophycocyanin, and CD38-PE-Cy7 (all from BD Biosciences).

Samples were measured on a FACSCanto flow cytometer. IgM and IgG levels were measured by

ELISA in culture supernatants as was described earlier32, using polyclonal rabbit anti-human IgG

and IgM reagents and a serum protein calibrator (all from Agilent Technologies, Santa Clara).

NK cell proliferation, cytokine production, and cytotoxicityHealthy donor PBMC were stimulated overnight with either a combination of IL-2 (100 U/ml,

Peprotech, Rocky Hill, NJ, USA) and IL-15 (10ng/ml, PeproTech), or a combination of IL-2, IL-12 (10

ug/ml, R&D Systems, Minneapolis, MN) and IL-18 (100 ug/ml, R&D Systems). PBMC fractions were

cocultured with K562 target cells (American Type Culture Collection, Manassas, VA) for 4 hours to

stimulate NK cells. Cells were stained with the following abs for flow cytometry: CD107a-PE-Cy7,

CD56-BUV395, CD3-V500, CD16-BV450, IFNγ-BV421, TNFα-BV650 and Granzyme B-Alexa Fluor

700 (all from BD Biosciences) and LIVE/DEAD Fixable Red Stain (Invitrogen). Intracellular stainings

were performed using Cytofix/Cytoperm reagent kit according to the manufacturer’s protocol

(BD Biosciences). For NK cell cytotoxicity, stimulated PBMC were cocultured with CellTrace Violet

(Invitrogen) labeled K562 target cells for 3 hours at a NK:K562 ratio of 1:1. Cell cultures were

labeled with MitoTracker Orange (Invitrogen) and TO-PRO-3 (Invitrogen) to determine target cell

death. For NK cell proliferation, PBMC were labeled with CFSE as described above. PBMC were

stimulated with IL-2 plus IL-15 or IL-2 plus IL-12 plus IL-18 for 5 days in the concentrations described

above. Fresh drugs were added after 3 days of stimulation. Samples were analyzed on an LSR

Fortessa (BD Biosciences), and data wre analyzed using FlowJo Version 10.

T cell stimulation using anti-CD3 and anti-CD28 AbsHealthy donor PBMC were labeled with CFSE, and stimulated with anti-CD3 (clone 1XE) and

anti-CD28 (clone 15E8) Abs for 4 days. Afterwards, cells were stained with CD3-Alexa Fluor

700 (ThermoFisher Scientific), CD4-PE-Cy7, CD8-PerCP-Cy5.5, CD25-allophycocyanin (all BD

Biosciences) and LIVE/DEAD Fixable Red Stain (Invitrogen). Samples were measured on FACSCanto

flow cytometer and data were analyzed using FlowJo Version 10. IFNγ production was measured

in the culture supernatant using human IFNγ Uncoated ELISA Kit, according to manufacturer’s

protocol (Thermo Fisher Scientific). Pure fractions of CD4 and CD8 T cells were isolated by positive

selection with MACS magnetic beads (Miltenyi Biotec, Bergish Gladbach, Germany) according to

the manufacturer’s instruction.

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Mixed lymphocyte reactionCD14+ cells were isolated by positive selection with MACS beads (Miltenyi Biotec) and differentiated

into monocyte-derived dendritic cells (moDCs) by incubation with IL-4 (20 ng/ml, R&D Systems)

and GM-CSF (1000 U/ml, Genzyme, Cambridge, MA) for 7 days, and maturated with LPS (100

ng/ml, Sigma-Aldrich) during the last 2 days. Healthy donor PBMC were labeled with CFSE, and

incubated with allogeneic moDCs for 4 days. Afterwards, analysis of T cell proliferation, activation

and IFNγ production by ELISA was performed as described above.

StatisticsTo visualize the relative effect of drug treatments on immune cell function, data of all experiments

wre normalized to the stimulated control without any drug added. Statistical analysis was

performed on raw data before normalization. Data were analyzed using repeated-measures one-

way ANOVA followed by a Dunnett’s multiple comparisons test. Statistical analysis was performed

using GraphPad Prism v7. Differences between groups were considered significant when p ≤ 0.05.

RESULTSJAK inhibitors block proliferation induced by CD40/IL-21, but do not induce cell death in CLL cellsWithin the microenvironment, CLL cells receive a variety of signals, including through cytokines,

BCR activation and costimulation via TNFR. The effects of kinase inhibition on microenvironmental

stimulation was tested using the JAK inhibitors momelotinib and ruxolitinib as well as with the BTK

inhibitor ibrutinib and PI3Kδ inhibitor idelalisib, currently used to treat CLL patients. Tirabrutinib

(GS4059) is a newly developed BTK inhibitor that is more selective compared to ibrutinib33,34.

Proliferation of CLL cells was induced by coculturing primary CLL cells on CD40L-expressing 3T3

fibroblasts in combination with IL-2112. Momelotinib was able to block IL-21 signaling and reduce

proliferation of CLL cells, to a greater extent than ruxolitinib (Figure 1A, Supplementary Figure

1A-C). As we have shown before, CD40L/IL-21 induced proliferation can be partially inhibited

by the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib19, and this also held for the BTK

inhibitor tirabrutinib. Momelotinib showed the strongest inhibition of proliferation of CLL cells.

JAK, BTK and PI3Kδ inhibitors did not induce cell death in CLL cells (Figure 1B). Cell death of CLL

cells can be induced by other therapeutic agents, such as the Bcl-2 inhibitor venetoclax, or the

chemotherapeutic agent fludarabine. We have previously demonstrated that CD40 stimulation,

as a model for lymph node prosurvival signals, renders CLL cells resistant to both venetoclax and

fludarabine35,36. As expected, JAK inhibitors were not able to reduce resistance to venetoclax

and fludarabine after coculture of CLL cells on CD40L-fibroblasts, because CD40 signaling is not

mediated by JAKs (Figure 1C).


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Figure 1. Eff ects of JAK inhibitors on CLL cells within the tumor microenvironment. The eff ect of JAK inhibitors on CLL signaling pathways within the tumor microenvironment. (A) CLL PBMC were cocultured on 3T40L cells and treated with IL-21 for 5 days to induce CLL cell proliferati on, in the presence of either JAK, BTK or PI3Kδ inhibitors (n=8). (B) CLL PBMC were sti mulated with IL-4 (24 hours) and anti -IgM (30 minutes) or medium control, with or without kinase inhibitors (n=3). Cell viability was measured by incubati ng cells with DiOC6 and PI (live cells defi ned as DiOC6+PI-). (C) CLL PBMC were cultured on 3T3 or 3T40L fi broblasts for 3 days in combinati on with JAK inhibitors. Aft erwards, cells were treated for 24 hours with either vene-toclax or fl udarabine (n=3). Viability was measured by incubati ng cells with DiOC6 and PI. (D) CLL cells were sti mulated with IL-4 and IgM in the presence of JAK, BTK or PI3Kδ inhibitors. Representati ve Western blot and quanti fi cati on showing phosphorylati on of target molecules of IL-4 and IgM signaling. LY294002 (1µM) and Rapamycin (1µM) were used as controls for PI3K and mTOR signaling, respecti vely. Bar graphs show summarized relati ve protein expression of 3 independent experiments. Bars indicate mean ± SD relati ve to conditi on without inhibitor, *p<0.05; **p<0.01, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).

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To study the effects of JAK inhibitors within the CLL microenvironment compared to other kinase

inhibitors, we studied their effects on signaling by IL-4 and IgM (a model for both T cell help and

BCR stimulation within the lymph node11) by Western blot. Treatment with both JAK inhibitors, but

not BTK or PI3Kδ inhibitors, led to a reduction in p-STAT6 induced by IL-4 (Figure 1D). Surprisingly,

momelotinib treatment also led to a reduction in IgM-induced p-Akt and p-S6 levels, although

not as strong as both BTK inhibitors or idelalisib. Ruxolitinib did not affect BCR signaling to Akt or

S6, demonstrating different modes of action of these JAK inhibitors. These results demonstrate

that JAK inhibitors are not cytotoxic for CLL cells by themselves, but are able to influence signaling

of prosurvival cytokines like IL-4 and IL-21 that induce proliferation and IgM expression, and

momelotinib was able to partially block BCR signaling.

JAK inhibition minimally affects healthy B cell functionNew small molecules used for CLL therapy can affect healthy B cells as well, especially the BTK

inhibitors. Proliferation of healthy B cells by stimulation with CpG/IL-2 was not inhibited by JAK

inhibitors (Figure 2A, Supplementary Figure 2A-C). In contrast, BTK and PI3Kδ inhibitors were

able to significantly reduce proliferation of healthy B cells. Similar to CLL B cells, JAK, BTK and

PI3Kδ inhibition did have an effect on proliferation of healthy B cells induced by CD40L and IL-21

stimulation (Supplementary Figure 2D). None of the kinase inhibitors significantly affected IgM

production after CpG/IL-2, although ibrutinib and idelalisib showed a clear trend of inhibition

(Figure 2B). A high dose of momelotinib was able to inhibit IgG production, in contrast to

ruxolitinib. As expected, both BTK inhibitors and idelalisib affected IgG production (Figure 2C).

Finally, differentiation of healthy B cells in response to CpG/IL-2 was not significantly altered by

JAK inhibitors (Figure 2D). BTK inhibition led to a slight reduction of B cell differentiation, whereas

PI3Kδ inhibition had no effect. These results indicate that JAK inhibition has only a moderate

effect on the function of healthy B cells, in contrast to BTK and PI3Kδ inhibitors, which inhibit the

function of healthy B cells to a greater extent.


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Figure 2. Eff ect of kinase inhibitors on healthy B cell functi on. Healthy donor PBMC were sti mulated with CpG and IL-2 for 6 days, in combinati on with JAK, BTK and PI3Kδ inhibitors (n=8 for all experiments). (A) Pro-liferati on of B cells aft er 6 day culture. (B) Levels of secreted IgM in culture supernatant measured by ELISA. (C) Levels of secreted IgG in culture supernatant measured by ELISA (D) Subset diff erenti ati on of B cells 6 days aft er sti mulati on. Representati ve examples of subset marker expression are shown in contour plots, quanti fi cati on of multi ple experiment in bar graphs. B cell subsets are defi ned as: naïve (IgD+CD27-), memory (IgD-CD27+) and plasmablast (CD27++CD38+). Bars indicate mean ± SD relati ve to conditi on without inhibitor, *p<0.05; **p<0.01, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).

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JAK inhibiti on ablates cytokine priming of NK cells, but not the acti vity via natural cytotoxicity receptorsAlthough NK cells do not require sti mulati on via cytokines, cytokines can increase the magnitude

of NK cell responses37. PBMC were sti mulated with either a combinati on of IL-2/IL-15 or IL-2/

IL-12/IL-18 to acti vate NK cells, and subsequently incubated with the classical NK target cell line

K562. IL-2/IL-15 sti mulati on induced NK cell proliferati on and increased eff ector responses of NK

cells toward K562 cells, resulti ng in increased producti on of IFNγ and TNFα, and higher levels of

degranulati on and target cell death (Figure 3A-E, Supplementary Figure 3A-C).

Figure 3. Eff ect of JAK inhibitors on NK cell functi on. Healthy donor PBMC were sti mulated with IL-2 and IL-15 for 5 days (A) or overnight (B-E) in combinati on with kinase inhibitors (n=8). (A) Proliferati on of NK cells aft er sti mulati on for 5 days. (B+C) Percentage of NK cells producing IFNγ (B) or TNFα (C) aft er 4 hour co-culture of sti mulated PBMC with K562 target cells as measured by fl ow cytometry. (D) Percentage of degranulated (CD107a+) NK cells aft er 4 hour co-culture with K562 target cells. (E) Specifi c lysis of K562 target cells aft er co-culture with sti mulated PBMC for 3 hours. Bars indicate mean ± SD relati ve to conditi on without inhibitor. *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).

Because both IL-2 and IL-15 signaling are dependent on JAK signaling, acti vati on via these cytokines

was effi ciently blocked by both JAK inhibitors, leading to a reducti on of NK responses. Ibruti nib

also showed a strong inhibitory eff ect on NK cell functi on aft er IL-2/IL-15 sti mulati on, especially

on the producti on of eff ector cytokines, whereas ti rabruti nib and idelalisib had smaller eff ects


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(Figure 3A-E). Stimulation with IL-2/IL-12/IL-18 also enhanced effector responses of NK cells

(Supplementary Figure 3D-H). NK cell proliferation upon IL-2/IL-12/IL-18 was blocked completely by

JAK inhibitors, whereas no or only partial effects on IL-2/IL-12/IL-18-enhanced cytokine production

and cytotoxicity were observed. Because IL-18 signaling is not JAK-dependent, but is a TLR-like

stimulus, it can be assumed that this stimulating signal is not affected by JAK inhibition, and NK

cells continue to receive an activating signal in this setting. Both BTK inhibitors and idelalisib had

only small effects on IL-2/IL-12/IL18 stimulation, although ibrutinib substantially affected NK cell

proliferation and TNFα production. Importantly, NK cell cytokine production and cytotoxicity in

response to target cells was not completely abrogated by JAK inhibitors, but returned to levels

similar to untreated PBMC, indicating that signaling via JAK-independent natural cytotoxicity

receptors is still functioning and that JAK inhibition only targets the cytokine signaling pathways.

JAK inhibition targets T cell activation and cytokine productionJAKs are involved in cytokine-induced amplification of T cell responses and differentiation

of specific T cell phenotypes. Stimulation of PBMC with anti-CD3 and anti-CD28 Abs induced

proliferation, activation, IFNγ production and T cell differentiation in both CD4 and CD8 T cells

(Figure 4A-F, Supplementary Figure 4A-D). Interestingly, ibrutinib showed the strongest inhibition

of T cell proliferation and activation. This was probably mediated via off-target inhibition of ITK26,27,

as the more selective tirabrutinib consistently showed less offtarget effects on T cell function at

any level. JAK inhibitors showed modest inhibition of CD8 T cell proliferation and activation, yet

the production of IFNγ was strongly inhibited, especially by momelotinib (Figure 4C+D). Although

results did not reach statistical significance because of patient variability, a clear relative effect

within donors was observed (Figure 4D). The inhibitory effects on IFNγ production of momelotinib,

ruxolitinib, ibrutinib and idelalisib are a direct effect on CD4 and CD8 T cells, as IFNγ production

by purified CD4 and CD8 T cell fractions was also inhibited (Figure 4E). Similar to the data in full

PBMC, CD8 T cells seemed more sensitive to JAK inhibitors compared with CD4 T cells. Although

idelalisib showed minimal effects on T cell activation and proliferation, it significantly affected

T cell differentiation after stimulation, leading to an increase in effector cell differentiation.

Conversely, ibrutinib had a small inhibitory effect on T cell differentiation (Figure 4F), whereas

JAK inhibition had no effect (Supplementary Figure 4C).

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Figure 4. Eff ect of kinase inhibitors on the functi on of healthy T cells. Healthy donor PBMC were sti mu-lated with anti -CD3 and anti -CD28 Abs for 4 days, in combinati on with JAK, BTK and PI3Kδ inhibitors (n=8). (A) Proliferati on of CD4 and CD8 T cells aft er sti mulati on for 4 days. (B) Expression of acti vati on marker CD25 on the surface of CD4 and CD8 T cells aft er 4 days of sti mulati on. (C) Levels of IFNγ in culture supernatants measured by ELISA. (D) Normalized values of panel C, showing a clear relati ve eff ect of most kinase inhibitors on IFNγ producti on of T cells. (E) IFNγ producti on of MACS purifi ed CD4 and CD8 T cell fracti ons aft er 4 days of sti mulati on (n=4). (F) T cell subset diff erenti ati on aft er 4 days of sti mulati on. T cell subsets are defi ned as: naïve (CD45RA+CD27+), Memory (CD45RA-CD27+), Eff ector (CD45RA-CD27-) and EMRA (CD45RA+CD27-). Bars indicate mean ± SD relati ve to conditi on without inhibitor (except for panel C, which depicts non-transformed data), *p<0.05; **p<0.01, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).


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JAK inhibitors and ibruti nib strongly aff ect allogeneic T cell responsesBecause both JAK inhibitors and ibruti nib have clinical acti vity in GvHD, we also tested the eff ects

of the inhibitors in a mixed lymphocyte reacti on. PBMC from healthy donors were mixed with

allogeneic LPS-maturated moDC, and T cell proliferati on, acti vati on and cytokine producti on were

determined aft er 4 days of coculture. Allogeneic T cell responses were signifi cantly inhibited by

both JAK inhibitors (especially momeloti nib) and ibruti nib (Figure 5A and B). Tirabruti nib showed

no inhibiti on of T cell functi on, in sharp contrast to ibruti nib, and in accordance with the response

to sti mulati on with anti -CD3 and anti -CD28 Abs. The inhibiti on of T cell acti vati on and proliferati on

by JAK inhibitors corresponded with a dosedependent decrease in IFNγ levels (Figure 5C). In

contrast, IFNγ levels consistently increased using the lower dose of ibruti nib and a similar trend

was seen with ti rabruti nib.

Figure 5. Kinase inhibitors modulate allogeneic T cell responses. Healthy donor PBMC were sti mulated with allogeneic LPS-maturated moDCs for 4 days, in combinati on with JAK, BTK and PI3Kδ inhibitors (n=8). (A) Proliferati on of CD4 and CD8 T cells aft er coculture for 4 days. (B) Expression of acti vati on marker CD25 on the surface of CD4 and CD8 T cells aft er 4 days of co-culture. (C) Levels of IFNγ in culture supernatants measured by ELISA. Bars indicate mean ± SD relati ve to conditi on without inhibitor, *p<0.05; **p<0.01, ***p<0.001, **** p<0.0001, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).

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DISCUSSIONIn the context of increasing application of kinase inhibitors in cancer treatment and emerging

awareness of infectious complications of these compounds, a thorough understanding of the

effects of JAK, BTK and PI3Kδ inhibitors on the function of both healthy and malignant lymphocytes

is warranted. JAK inhibitors could play a beneficial role for the treatment of CLL, by blocking

signaling of important prosurvival molecules like IL-4 and IL-21. Although our results indicate that

JAK inhibitors do not have detrimental effects on the function of healthy B cells (in contrast to BTK

and PI3Kδ inhibitors), the observed inhibition of NK and T cell function could have significant side

effects during patient treatment. Off-target effects on T cell function also occurs with ibrutinib,

leading to functional impairments, but these effects are not observed with the more selective

BTK inhibitor tirabrutinib.

We studied the effect of kinase inhibitors on healthy and malignant B cell function. Although

momelotinib and ruxolitinib are both JAK1/2 inhibitors, we observe clear differential effects on BCR

signaling. In particular, momelotinib inhibits BCR mediated phosphorylation of Akt and S6, whereas

ruxolitinib did not target BCR signaling. The inhibition of BCR signaling by momelotinib might be

beneficial during CLL therapy, as the BCR pathway plays an important role in the pathology of

the disease11. Recently, a role for JAK2 has been described in BTK activation, especially in the

context of CXCR4 signaling38. If the effects of momelotinib on BCR signaling are via on-target JAK2

inhibition, it is unclear why ruxolitinib does not induce similar effects. Therefore, differential off-

target effects of momelotinib and ruxolitinib might explain our observations. Momelotinib also

inhibits TANK-binding kinase 1 (TBK1) and IKKε, kinases that are involved in NF-κB signaling upon

activation by TLR and RIG1 signaling, suggesting that these kinases play a role in the inhibitory

effect of momelotinib on BCR signaling39.

Off-target effects of kinase inhibitors have been described, e.g. the observed effect of ibrutinib on

T cell function is in line with reports on off-target ITK inhibition26,27. More specific BTK inhibitors,

like tirabrutinib, have been developed to limit offtarget effects33,34. Tirabrutinib showed similar

inhibition of B cell function, in both healthy B cells and CLL cells, but no inhibition of T cell function.

Allogeneic T cell responses were also inhibited by ibrutinib, but not tirabrutinib. Surprisingly, IFNγ

levels increased in mixed lymphocyte reactions with both inhibitors, suggesting a BTK-mediated

effect that is perhaps explained by BTK-dependent improvement of the Ag presenting capacity of

dendritic cells40. The disappearance of this effect with a higher dose of ibrutinib may well reflect

ITK inhibition.

Tirabrutinib may, therefore, have less infectious complications compared with ibrutinib. Together

with promising phase 1 trial results33,34 these data support further exploration of tirabrutinib

or other more selective BTK inhibitors as a clinical treatment in CLL and other malignancies. In

contrast, offtarget effects of ibrutinib on T cells are not always detrimental, as has been observed

in CLL, in which ibrutinib treatment increases T cell numbers and improves the efficacy of chimeric

antigen receptor T cell therapy27,41.


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Several prosurvival stimuli that CLL cells receive from the microenvironment are mediated via

JAK signaling, like T cell derived IL-4 and IL-2111,12. In CLL patients, ruxolitinib leads to lymphocyte

redistribution out of the lymph nodes and disease stabilization, establishing in vivo activity of

JAK inhibition20,21. Although the efficacy of ruxolitinib monotherapy in patients unfit for regular

chemo-immunotherapy is only moderate, the egress of tumor cells from lymph nodes observed

with ruxolitinib treatment is similar to the lymphocytosis seen during the early phases of ibrutinib

or idelalisib treatment20,21. JAK inhibitors may therefore be useful compounds in combination

with cell death-inducing agents such as the Bcl-2 inhibitor venetoclax, even in patients refractory

to ibrutinib or idelalisib, by depriving tumor cells of microenvironmental stimuli. Because

combination treatment with venetoclax and ibrutinib has shown promising clinical efficacy in

CLL42,43, combinatory treatment of JAK inhibitors and venetoclax may pose similar clinical benefit.

The strong inhibitory effects on T and NK cell function by JAK inhibitors observed by us and

others23,24 may complicate their use in CLL. Ruxolitinib treatment is associated with an increased

risk of infectious complications, predominantly with infections of the urogenital and respiratory

tract, but reactivation of hepatitis B and tuberculosis also occurs44. The increased risk of infections

may pose a significant problem for already frail CLL patients, where infections are a leading cause

of death1. Indeed, ruxolitinib treatment led to infectious complications in unfit CLL patients21.

The functional impairment of lymphocytes by JAK inhibitors can be beneficial in other disease

settings, such as autoimmune diseases and GvHD. Both T and B cells are implicated in GvHD

pathology45 and ruxolitinib has clinical efficacy in refractory GvHD patients46. However, we show

in this study that JAK inhibitors have relatively mild effects on B cell function, which may indicate

that pathogenic B cell responses remain relatively intact in GvHD during ruxolitinib treatment.

The inhibition of both T and B cell function provides a rationale to use ibrutinib for GvHD therapy.

Indeed, our data demonstrate that ibrutinib also inhibits T cells responses toward allogeneic cells.

In clinical trials, ibrutinib treatment led to clinical responses in two-thirds of refractory GvHD

patients, with efficacy in all affected organs and a reduction in steroid use, and ibrutinib is now a

Food and Drug Administration approved drug for glucocorticoid-resistant GvHD47.

In conclusion, we show that JAK inhibitors potently inhibit several prosurvival stimuli for CLL

cells. However, their inhibition of T and NK cell function, and consequently the increased risk of

infections, may complicate clinical treatment with JAK inhibitors in CLL patients. The BTK inhibitor

tirabrutinib consistently showed a similar inhibitory potential as ibrutinib, but lacked the offtarget

effects of ibrutinib on T cells, which warrants future research comparing tirabrutinib with ibrutinib

in the clinical setting. Conversely, the inhibition of T and NK cell function we observe by JAK

inhibitors and ibrutinib can be beneficial in disease settings with unwanted lymphocyte activation,

such as GvHD. Our data are in line with early clinical data and demonstrate that kinase inhibitors

in development as antitumor drugs in hematological malignancies can also be applied to block

unwanted lymphocyte function in other diseases. The properties of individual kinase inhibitors

can be exploited in combination treatment strategies tailored to the inhibitory effects desired.

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SUPPLEMENTARY DATA

Supplementary Figure 1. Proliferati on of CLL cells aft er treatment with kinase inhibitors. Proliferati on of CLL PBMC was induced by coculturing on 3T40L cells for 5 days while sti mulati ng with IL-21. (A) Gati ng strategy showing from left to right the gati ng of lymphocytes, viable cells, and CD19+CD5+ CLL cells. (B) Representati ve histograms showing the proliferati on of CLL PBMC aft er 5 days in the unsti mulated conditi on, the untreated conditi on, and the eff ects of kinase inhibitors on proliferati on. (C) Non-transformed proliferati on data of CLL cells aft er CD40L plus IL-21 sti mulati on and the infl uence of kinase inhibitors, corresponding to Figure 1A.

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Supplementary Figure 2. Proliferati on of healthy donor B cells aft er CpG and IL-2 sti mulati on. (A) Gati ng strategy for healthy B cells aft er CpG and IL-2 sti mulati on. From left to right, samples were gated on lym-phocytes, single cells, viable cells, and fi nally CD19+ B cells. (B) Representati ve histograms of proliferati on of healthy donor B cells aft er sti mulati on with CpG and IL-2 for 6 days, with and without kinase inhibitor treat-ment. (C) Non-transformed proliferati on data of healthy B cells aft er CpG and IL-2 sti mulati on, corresponding to Figure 2A (n=8). (D) Proliferati on of healthy B cells aft er sti mulati on with CD40L plus IL-21 for 6 days, and the eff ect of kinase inhibitors (n=4). (E) Viability of healthy B cells aft er 6 days of sti mulati on and treatment with kinase inhibitors. Bars indicate mean ± SD. *p<0.05; **p<0.01, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).


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Supplementary Figure 3. Infl uence of kinase inhibitors on IL-2 plus IL-12 plus IL-18 sti mulati on of NK cells. (A) Representati ve histograms of NK cell proliferati on aft er sti mulati on with IL-2 and IL-15 for 5 days, and the infl uence of kinase inhibitors. (B) Viability of NK cells aft er 5 days of IL-2 plus IL-15 sti mulati on in the presence of kinase inhibitors. (C) Gati ng strategy of IFNγ and TNFα producing, and degranulati ng (CD107a+) NK cells aft er sti mulati on with K562 cells. (D) Proliferati on of NK cells aft er sti mulati on with IL-2 plus IL-12 plus IL-18 for 5 days (n=8). (E+F) Percentage of NK cells producing IFNγ (E) or TNFα (F) aft er coculture of IL-2 plus IL-12 plus IL-18 sti mulated PBMC with K562 target cells (n=8). (G) Percentage of degranulated (CD107a+) NK cells aft er coculture with K562 target cells (n=8). (H) Specifi c lysis of K562 target cells aft er co-culture with IL-2 plus IL-12 plus IL-18 sti mulated PBMC for 3 hours (n=4). Bars indicate mean ± SD relati ve to conditi on without inhibitor, *p< 0.05; **p<0.01, ***p<0.001, ****p<0.0001, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test (stati sti cs were performed on non-transformed data).

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Supplementary Figure 4. Eff ect of kinase inhibitors on proliferati on, acti vati on and diff erenti ati on of T cells aft er anti -CD3 and anti -CD28 sti mulati on. (A) Representati ve histograms of proliferati on of healthy donor CD4 and CD8 T cells aft er anti -CD3 and anti -CD28 sti mulati on for 4 days. (B) Acti vati on of healthy donor CD4 and CD8 T cells aft er anti -CD3 and anti -CD28 sti mulati on for 4 days. (C) Eff ect of momeloti nib and ruxoliti nib on T cell diff erenti ati on during acti vati on (n=8). (D) Viability of CD4 and CD8 T cells aft er 4 days of sti mulati on in the presence of kinase inhibitors. Bars indicate mean ± SD relati ve to conditi on without inhibitor, *p<0.05, repeated measures one-way ANOVA followed by Dunnett ’s multi ple comparisons test.


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