Dissection of the Effects of JAK and BTK Inhibitors on the Functionality of Healthy and Malignant Lymphocytes

This information is current as Tom Hofland, Iris de Weerdt, Hanneke ter Burg, Renate de of October 1, 2021. Boer, Stacey Tannheimer, Sanne H. Tonino, Arnon P. Kater and Eric Eldering J Immunol published online 11 September 2019 http://www.jimmunol.org/content/early/2019/09/10/jimmun ol.1900321 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 11, 2019, doi:10.4049/jimmunol.1900321 The Journal of Immunology

Dissection of the Effects of JAK and BTK Inhibitors on the Functionality of Healthy and Malignant Lymphocytes

Tom Hofland,*,†,1 Iris de Weerdt,*,†,1 Hanneke ter Burg,*,† Renate de Boer,*,† Stacey Tannheimer,‡ Sanne H. Tonino,†,x Arnon P. Kater,†,x and Eric Eldering*,x

Despite the emergence of small molecule inhibitors, current treatment strategies for chronic lymphocytic (CLL) are not curative, and the search for new therapeutic modalities continues. Prosurvival signaling derived from the microenvironment 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 because of the effect on other immune cells, a feature shared with other kinase inhibitors used for CLL treatment, such as the BTK inhibitor and the PI3Kd inhibitor idelalisib. We compared functional effects of the JAK1/2 inhibitors and , the BTK inhibitors ibrutinib and tirabrutinib, Downloaded from and PI3Kd inhibitor idelalisib on malignant CLL cells but also on 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 -induced proliferation of CLL cells. Momelotinib also reduced 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 because of ITK inhibition. Remarkably, both BTK inhibitors stimulated IFN-g production in a mixed lymphocyte reaction. Collectively, our results demonstrate that kinase inhibitors directed at identical targets may have differential http://www.jimmunol.org/ effects on lymphocyte function. Their unique profile could be strategically employed to balance desired versus unwanted lymphocyte inhibition. The Journal of Immunology, 2019, 203: 000–000.

hronic lymphocytic leukemia (CLL) is characterized by receive prosurvival signaling from surrounding stromal cells, T cells, the accumulation of malignant B cells in blood, lymph and macrophages (1, 8–10). Therefore, disrupting prosurvival sig- C nodes, and bone marrow (1). Despite the development of naling within the microenvironment is a sensible therapeutic aim in targeted compounds and to eradicate CLL cells, CLL treatment. T cell–derived IL-4 and IL-21 are known survival none of the current treatments for CLL is curative. The Bcl-2 factors that both signal via JAK (11, 12). Inhibitors of JAKs have by guest on October 1, 2021 inhibitor and Bruton tyrosine kinase (BTK) inhibitor been developed, most notably momelotinib and ruxolitinib (both ibrutinib have shown substantial clinical efficacy in most CLL targeting JAK1 and JAK2). Ruxolitinib has been approved by the patients (2, 3). However, mutations leading to drug resistance have Food and Drug Administration and European Medicines Agency already been described for both drugs, and, because of the high for the treatment of myelofibrosis. Therapeutic inhibition of economic burden of lifelong treatments, alternative strategies JAKs with these inhibitors has shown significant clinical re- leading to actual curative treatments should still be pursued (4–7). sponse in myelofibrosis patients, leading to reduced spleen sizes Most treatments in CLL are counteracted by the tumor-supportive and improved overall survival (13–16). More recently, JAK in- microenvironment in secondary lymphoid organs, where CLL cells hibitors have also shown promising clinical results in graft- versus-host disease (GvHD), leading to a reduction of steroid need (17, 18). In CLL, the biological activity of JAK inhibitors is *Department of Experimental Immunology, Amsterdam Infection and Immunity demonstrated by lymphocyte redistribution out of the lymph Institute, Amsterdam University Medical Centers, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands; †Department of Hematology, Cancer Center nodes and disease stabilization. Although JAK inhibitors lack Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, efficacy as monotherapeutic agents, based on the prosurvival 1105 AZ Amsterdam, the Netherlands; ‡Gilead Sciences, Foster City, CA 94404; x contribution of IL-4 and IL-21, combination strategies involving and and Myeloma Center 1105 AZ Amsterdam, Amsterdam, the Netherlands JAK inhibitors may improve clinical responses in CLL treatment 1T.H. and I.d.W. contributed equally. (19–21). As JAK signaling plays a central role in the function of many Received for publication March 18, 2019. Accepted for publication August 10, 2019. immune cells, it is not surprising that JAK inhibitors have been Address correspondence and reprint requests to Tom Hofland or Prof. Eric Eldering, Department of Experimental Immunology, Hematology, Amsterdam University Med- shown to modulate the function of T cells, NK cells, and dendritic ical Centers, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands (T.H.) or De- cells (22–25). In addition, other kinase inhibitors currently used partment of Experimental Immunology, Lymphoma and Myeloma Center, Academic or studied for CLL treatment also display side effects by the Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands (E.E.). E-mail addresses: t.hofl[email protected] (T.H.) or [email protected] (E.E.) inhibition of off-target kinases. For example, ibrutinib has well- The online version of this article contains supplemental material. documented off-target effects on T cells by binding to IL-2– Abbreviations used in this article: Amsterdam UMC, Amsterdam University Medical inducible tyrosine kinase (ITK) (26, 27). Idelalisib, a PI3Kd Centers; BTK, Bruton tyrosine kinase; CLL, chronic lymphocytic leukemia; GvHD, inhibitor used to treat refractory CLL patients, alters T cell function graft-versus-host disease; ITK, inducible tyrosine kinase; moDC, monocyte-derived through modulation of TCR signaling, possibly explaining the in- dendritic cell; PI, propidium iodide. creased amount of atypical infections and autoimmune complica- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 tions observed in patients treated with idelalisib (28–30). To assess

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900321 2 DIFFERENTIAL EFFECTS OF KINASE INHIBITORS ON LYMPHOCYTES the clinical potential of a kinase inhibitor for CLL therapy, the Biosciences). Samples were measured on a FACSCanto flow cytometer. combined on- and off-target effects on both malignant and healthy IgM and IgG levels were measured by ELISA in culture supernatants, as cells need to be taken into account. In this study, we perform was described earlier (32), using polyclonal rabbit anti-human IgG and IgM reagents and a serum protein calibrator (all from Agilent Technolo- comparative studies of the effects of JAK, BTK, and PI3Kd in- gies, Santa Clara, CA). hibitors on CLL cells and healthy immune cells. We determine both beneficial and detrimental effects of all kinase inhibitors and ex- NK cell proliferation, cytokine production, and cytotoxicity plore whether therapeutic rationales exist to use combinations of Healthy donor PBMC were stimulated overnight with either a combination these inhibitors not only for CLL therapy but also for other diseases. of IL-2 (100 U/ml; PeproTech, Rocky Hill, NJ) and IL-15 (10 ng/ml; PeproTech) or a combination of IL-2, IL-12 (10 mg/ml; R&D Systems, Minneapolis, MN), and IL-18 (100 mg/ml; R&D Systems). PBMC frac- Materials and Methods tions were cocultured with K562 target cells (American Type Culture Patient and healthy donor samples Collection, Manassas, VA) for 4 h to stimulate NK cells. Cells were stained with the following Abs for flow cytometry: CD107a-PE-Cy7, CD56– Peripheral blood samples from untreated CLL patients were collected at BUV395, CD3–V500, CD16–BV450, IFN-g–BV421, TNF-a–BV650, and the Amsterdam University Medical Centers (Amsterdam UMC), Aca- granzyme B–Alexa Fluor 700 (all BD from Biosciences) and LIVE/DEAD demic Medical Center in Amsterdam, the Netherlands. Healthy donor Fixable Red Stain (Invitrogen). Intracellular stainings were performed PBMC were isolated from buffy coats obtained from Sanquin using Cytofix/Cytoperm reagent according to the manufacturer’s pro- Blood Supply (Amsterdam, the Netherlands). Ethical approval was tocol (BD Biosciences). For NK cell cytotoxicity, stimulated PBMC were provided by the medical ethical committee at the Amsterdam UMC, cocultured with CellTrace Violet (Invitrogen)–labeled K562 target cells Academic Medical Center, and written informed consent was obtained for 3 h in a NK:K562 at a ratio of 1:1. Cell cultures were labeled with in accordance with the Declaration of Helsinki. PBMC from CLL MitoTracker Orange (Invitrogen) and TO-PRO-3 (Invitrogen) to determine patients and healthy donors were isolated and cryopreserved as de- target cell death. For NK cell proliferation, PBMC were labeled with CFSE Downloaded from scribed earlier (31). as described above. PBMC were stimulated with IL-2 plus IL-15 or IL-2 plus IL-12 plus IL-18 for 5 d in the concentrations described above. Cell lines Freshdrugswereaddedafter3dofstimulation. Samples were analyzed on an LSRFortessa (BD Biosciences), and data were analyzed using NIH-3T3 fibroblasts (ACC number 59; Deutsche Sammlung von FlowJo Version 10. Mikroorganismen und Zellkulturen, Braunschweig, Germany) and 3T40L (NIH-3T3 fibroblasts expressing CD40L) were cultured in IMDM (Thermo T cell stimulation using anti-CD3 and anti-CD28 Abs

Fisher Scientific, Waltham, MA) supplemented with 10% FCS and 1% http://www.jimmunol.org/ penicillin/streptomycin. Healthy donor PBMC were labeled with CFSE and stimulated with anti- CD3 (clone 1XE) and anti-CD28 (clone 15E8) Abs for 4 d. Afterwards, Compounds cells were stained with CD3–Alexa Fluor 700 (Thermo Fisher Scientific), CD4-PE-Cy7, CD8-PerCP-Cy5.5, CD25–allophycocyanin (all BD Bio- The JAK inhibitors momelotinib and ruxolitinib, the BTK inhibitor tirabrutinib, sciences), and LIVE/DEAD Fixable Red Stain (Invitrogen). Samples were and the PI3Kd inhibitor idelalisib were all obtained from Gilead Sciences measured on a FACSCanto flow cytometer, and data were analyzed using (Foster City, CA). The BTK inhibitor ibrutinib was purchased from FlowJo Version 10. IFN-g production was measured in the culture super- Selleckchem (Houston, TX). Venetoclax (ABT-199) was purchased from natant using a human IFN-g Uncoated ELISA Kit, according to manufac- Sanbio (Uden, the Netherlands). Fludarabine was purchased from Sigma- turer’s protocol (Thermo Fisher Scientific). Pure fractions of CD4 and CD8 Aldrich (St Louis, MO). All compounds were used in concentrations T cells were isolated by positive selection with MACS magnetic beads close to their EC values or clinically relevant levels. 50 (Miltenyi Biotec, Bergish Gladbach, Germany) according to manufacturer’s by guest on October 1, 2021 Proliferation and chemoresistance of CLL cells instruction. For proliferation experiments, PBMC from CLL patients were labeled with Mixed lymphocyte reaction 0.5 mM CFSE (Thermo Fisher Scientific) and cultured for 5 d on either CD14+ cells were isolated by positive selection with MACS beads (Mil- NIH-3T3 or 3T40L cells with or without 25 ng/ml rIL-21 (Invitrogen, tenyi Biotec) and differentiated into monocyte-derived dendritic cells Carlsbad, CA). Cultures were measured on a FACSCanto (BD Biosciences, (moDCs) by incubation with IL-4 (20 ng/ml; R&D Systems) and GM-CSF San Jose, CA), and data were analyzed using FlowJo Version 10. To study (1000 U/ml; Genzyme, Cambridge, MA) for 7 d and maturated with LPS chemoresistance of CLL cells, PBMC from CLL patients were cocultured (100 ng/ml; Sigma-Aldrich) during the last 2 d. Healthy donor PBMC were with NIH-3T3 or 3T40L cells for 3 d. Cells were treated with venetoclax or labeled with CFSE and incubated with allogeneic moDCs for 4 d. After- fludarabine for 24 h. Target cell death was analyzed by incubating cultures wards, analysis of T cell proliferation, activation, and IFN-g production by with DiOC6 (Thermo Fisher Scientific) and propidium iodide (PI) (Sigma- ELISA was performed as described above. Aldrich). Cells were analyzed on a FACSCanto flow cytometer. Data were analyzed using FlowJo Version 10. Statistics CLL cell viability and Western blot analysis To visualize the relative effect of drug treatments on immune cell function, data of all experiments were normalized to the stimulated control without any CLL-derived PBMC were preincubated for 1 h with inhibitors and stim- drug added. Statistical analysis was performed on raw data before normali- ulated for 24 h with IL-4 (10 ng/ml; Bio-Techne, Minneapolis, MN) and zation. Data were analyzed using repeated measures one-way ANOVAfollowed during the last 30 min with anti-IgM (20 mg/ml; BioLegend, San Diego, by a Dunnett multiple comparisons test. Statistical analysis was per- CA). Viability was measured by FACS using DiOC6/PI staining as de- formed using GraphPad Prism v7. Differences between groups were scribed above. For Western blot, cell lysates were prepared by lysing in considered significant when p , 0.05. 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 s of soni- cation in a Branson sonicator (Danbury, CT). Lysates were analyzed by Results SDS-PAGE. Western blot was performed using the following Abs: rabbit JAK inhibitors block proliferation induced by CD40/IL-21 but anti–p-STAT6, rabbit anti-pErk, rabbit anti-pAkt (Ser473), rabbit anti-pS6, do not induce cell death in CLL cells mouse anti-S6 (all from Cell Signaling Technology [Danvers, MA]) and goat anti-actin (Santa Cruz Biotechnology, Dallas, TX). Within the microenvironment, CLL cells receive a variety of Proliferation, IgM/IgG production and differentiation of signals, including through , BCR activation, and healthy B cells costimulation via TNFR. The effects of kinase inhibition on microenvironmental stimulation was tested using the JAK in- Healthy donor PBMC were stained with CFSE, and stimulated with CpG hibitors momelotinib and ruxolitinib as well as the BTK in- ODN2006 (1 mg/ml; Invitrogen) and IL-2 (100 U/ml) for 6 d. Fresh drugs were added after 3 d of stimulation. Cells were stained with the following hibitor ibrutinib and the PI3Kd inhibitor idelalisib, currently Abs for flow cytometry: CD19-PerCP-Cy5.5, CD20- allophycocyanin-H7, used to treat CLL patients. Tirabrutinib (GS4059) is a newly IgD–PE, CD27–allophycocyanin, and CD38-PE-Cy7 (all from BD developed BTK inhibitor that is more selective compared with The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Effects of JAK inhibitors on CLL cells within the tumor microenvironment. The effect 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 d to induce CLL cell proliferation in the presence of either JAK, BTK, or PI3Kd inhibitors (n = 8). (B) CLL PBMC were stimulated with IL-4 (24 h) and anti-IgM (30 min) or medium control, with or without kinase inhibitors (n = 3). Cell viability was measured by incubating cells with DiOC6 and PI (live cells defined as DiOC6+PI2). (C) CLL PBMC were cultured on 3T3 or 3T40L fibroblasts for 3 d in combination with JAK inhibitors. Afterwards, cells were treated for 24 h with either venetoclax or fludarabine (n = 3). Viability was measured by incubating cells with DiOC6 and PI. (D) CLL cells were stimulated with IL-4 (Figure legend continues) 4 DIFFERENTIAL EFFECTS OF KINASE INHIBITORS ON LYMPHOCYTES ibrutinib (33, 34). Proliferation of CLL cells was induced by JAK inhibition ablates cytokine priming of NK cells but not the coculturing primary CLL cells on CD40L-expressing 3T3 fibro- activity via natural cytotoxicity receptors blasts in combination with IL-21 (12). Momelotinib was able to Although NK cells do not require stimulation via cytokines, cy- block IL-21 signaling and reduce proliferation of CLL cells to a tokines can increase the magnitude of NK cell responses (37). greater extent than ruxolitinib (Fig. 1A, Supplemental Fig. 1A–C). PBMC were stimulated with either a combination of IL-2/IL-15 As we have shown before, CD40L/IL-21–induced proliferation can or IL-2/IL-12/IL-18 to activate NK cells and, subsequently, in- be partially inhibited by the BTK inhibitors ibrutinib and the PI3Kd cubated with the classical NK target cell line K562. IL-2/IL-15 inhibitor idelalisib (19), and this also held for the BTK inhibitor stimulation induced NK cell proliferation and increased effector tirabrutinib. Momelotinib showed the strongest inhibition of responses of NK cells toward K562 cells, resulting in increased proliferation of CLL cells. JAK, BTK, and PI3Kd inhibitors did production of IFN-g, TNF-a, and higher levels of degranulation not induce cell death in CLL cells (Fig. 1B). Cell death of CLL and target cell death (Fig. 3A–E, Supplemental Fig. 3A–C). cells can be induced by other therapeutic agents, such as the Bcl- Because both IL-2 and IL-15 signaling are dependent on JAK 2 inhibitor venetoclax or the chemotherapeutic agent fludar- signaling, activation via these cytokines was efficiently blocked abine. We have previously demonstrated that CD40 stimulation, by both JAK inhibitors, leading to a reduction of NK responses. as a model for lymph node prosurvival signals, renders CLL cells Ibrutinib also showed a strong inhibitory effect on NK cell resistant to both venetoclax and fludarabine (35, 36). As ex- function after IL-2/IL-15 stimulation, especially on the produc- pected, JAK inhibitors were not able to reduce resistance to tion of effector cytokines, whereas tirabrutinib and idelalisib had venetoclax and fludarabine after coculture of CLL cells on smaller effects (Fig. 3A–E). Stimulation with IL-2/IL-12/IL-18 CD40L-fibroblasts because CD40 signaling is not mediated by

also enhanced effector responses of NK cells (Supplemental Fig. Downloaded from JAKs (Fig. 1C). To study the effects of JAK inhibitors within the 3D–H). NK cell proliferation upon IL-2/IL-12/IL-18 was blocked CLL microenvironment compared with other kinase inhibitors, completely by JAK inhibitors, whereas no or only partial effects on we studied their effects on signaling by IL-4 and IgM [a model IL-2/IL-12/IL-18–enhanced cytokine production and cytotoxicity for both T cell help and BCR stimulation within the lymph node were observed. Because IL-18 signaling is not JAK dependent but is (11)] by Western blot. Treatment with both JAK inhibitors but a TLR-like stimulus, it can be assumed that this stimulating signal is not BTK or PI3Kd inhibitors led to a reduction in p-STAT6 in- not affected by JAK inhibition, and NK cells continue to receive an http://www.jimmunol.org/ duced by IL-4 (Fig. 1D). Surprisingly, momelotinib treatment activating signal in this setting. Both BTK inhibitors and idelalisib had also led to a reduction in IgM-induced p-Akt and p-S6 levels, only small effects on IL-2/IL-12/IL-18 stimulation, although ibrutinib although not as strong as both BTK inhibitors or idelalisib. substantially affected NK cell proliferation and TNF-a production. Ruxolitinib did not affect BCR signaling to Akt or S6, demon- Importantly, NK cell cytokine production and cytotoxicity in response strating different modes of action of these JAK inhibitors. These to target cells was not completely abrogated by JAK inhibitors but results demonstrate that JAK inhibitors are not cytotoxic for CLL returned to levels similar to untreated PBMC, indicating that signaling cells by themselves, but are able to influence signaling of pro- via JAK-independent natural cytotoxicity receptors is still functioning survival cytokines like IL-4 and IL-21 that induce proliferation and that JAK inhibition only targets the cytokine signaling pathways. and IgM expression, and momelotinib was able to partially block by guest on October 1, 2021 BCR signaling. JAK inhibition targets T cell activation and cytokine production JAK inhibition minimally affects healthy B cell function JAKs are involved in cytokine-induced amplification of T cell re- New small molecules used for CLL therapy can affect healthy sponses and differentiation of specific T cell phenotypes. Stimulation B cells as well, especially the BTK inhibitors. Proliferation of of PBMC with anti-CD3 and anti-CD28 Abs induced prolifera- healthy B cells by stimulation with CpG/IL-2 was not inhibited by tion, activation, IFN-g production, and T cell differentiation JAK inhibitors (Fig. 2A, Supplemental Fig. 2A–C). In contrast, in both CD4 and CD8 T cells (Fig. 4A–F, Supplemental Fig. 4A–D). BTK and PI3Kd inhibitors were able to significantly reduce Interestingly, ibrutinib showed the strongest inhibition of T cell proliferation of healthy B cells. Similar to CLL B cells, JAK, proliferation and activation. This was probably mediated via off- BTK, and PI3Kd inhibition did have an effect on proliferation target inhibition of ITK (26, 27), as the more selective tirabrutinib of healthy B cells induced by CD40L and IL-21 stimulation consistently showed less off-target effects on T cell function at any (Supplemental Fig. 2D). None of the kinase inhibitors signifi- level. JAK inhibitors showed modest inhibition of CD8 T cell cantly affected IgM production after CpG/IL-2, although proliferation and activation, yet the production of IFN-g was ibrutinib and idelalisib showed a clear trend of inhibition strongly inhibited, especially by momelotinib (Fig. 4C, 4D). (Fig. 2B). A high dose of momelotinib was able to inhibit IgG Although results did not reach statistical significance because of production, in contrast to ruxolitinib. As expected, both BTK patient variability, a clear relative effect within donors was ob- inhibitors and idelalisib affected IgG production (Fig. 2C). served (Fig. 4D). The inhibitory effects on IFN-g production of Finally, differentiation of healthy B cells in response to CpG/ momelotinib, ruxolitinib, ibrutinib, and idelalisib are a direct IL-2 was not significantly altered by JAK inhibitors (Fig. 2D). effect on CD4 and CD8 T cells, as IFN-g production by pu- BTK inhibition led to a slight reduction of B cell differentia- rified CD4 and CD8 T cell fractions was also inhibited (Fig. tion, whereas PI3Kd inhibition had no effect. These results 4E). Similar to the data in full PBMC, CD8 T cells seemed indicate that JAK inhibition has only a moderate effect on the more sensitive to JAK inhibitors compared with CD4 T cells. function of healthy B cells, in contrast to BTK and PI3Kd inhibi- Although idelalisib showed minimal effects on T cell activation tors, which inhibit the function of healthy B cells to a greater extent. and proliferation, it significantly affected T cell differentiation and IgM in the presence of JAK, BTK, or PI3Kd inhibitors. Representative Western blot and quantification showing phosphorylation of target molecules of IL-4 and IgM signaling. LY294002 (1 mM) and rapamycin (1 mM) were used as controls for PI3K and mTOR signaling, respectively. Bar graphs show summarized relative protein expression of three independent experiments. Bars indicate mean 6 SD relative to condition without inhibitor. *p , 0.05, **p , 0.01, repeated measures one-way ANOVA followed by Dunnett multiple comparisons test (statistics were performed on nontransformed data). The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Effect of kinase inhibitors on healthy B cell function. Healthy donor PBMC were stimulated with CpG and IL-2 for 6 d in combination with JAK, BTK, and PI3Kd inhibitors (n = 8 for all experiments). (A) Proliferation of B cells after 6 d 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 differentiation of B cells 6 d after stimulation. Representative examples of subset marker expression is shown in contour plots, quantification of multiple experiments are (Figure legend continues) 6 DIFFERENTIAL EFFECTS OF KINASE INHIBITORS ON LYMPHOCYTES Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Effect of JAK inhibitors on NK cell function. Healthy donor PBMC were stimulated with IL-2 and IL-15 for 5 d (A) or overnight (B–E)in combination with kinase inhibitors (n = 8). (A) Proliferation of NK cells after stimulation for 5 d. (B and C) Percentage of NK cells producing IFN-g (B)or TNF-a (C) after 4 h coculture of stimulated PBMC with K562 target cells, as measured by flow cytometry. (D) Percentage of degranulated (CD107a+)NK cells after 4 h coculture with K562 target cells. (E) Specific lysis of K562 target cells after coculture with stimulated PBMC for 3 h. Bars indicate mean 6 SD relative to condition without inhibitor. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, repeated measures one-way ANOVA followed by Dunnett multiple comparisons test (statistics were performed on nontransformed data). after stimulation, leading to an increase in effector cell differ- JAK inhibitors (especially momelotinib) and ibrutinib (Fig. 5A, 5B). entiation. Conversely, ibrutinib had a small inhibitory effect on Tirabrutinib showed no inhibition of T cell function, in sharp con- T cell differentiation (Fig. 4F), whereas JAK inhibition had no trast to ibrutinib and in accordance with the response to stimulation effect (Supplemental Fig. 4C). with anti-CD3 and anti-CD28 Abs. The inhibition of T cell activa- tion and proliferation by JAK inhibitors corresponded with a dose- JAK inhibitors and ibrutinib strongly affect allogeneic dependent decrease in IFN-g levels (Fig. 5C). In contrast, IFN-g T cell responses levels consistently increased using the lower dose of ibrutinib, and a Because both JAK inhibitors and ibrutinib have clinical activity in similar trend was seen with tirabrutinib. GvHD, we also tested the effects of the inhibitors in a mixed lymphocyte reaction. PBMC from healthy donors were mixed with allogeneic LPS-maturated moDC, and T cell proliferation, activa- Discussion tion, and cytokine production were determined after 4 d of coculture. In the context of increasing application of kinase inhibitors in can- Allogeneic T cell responses were significantly inhibited by both cer treatment and emerging awareness of infectious complications of shown in bar graphs. B cell subsets are defined as follows: naive (IgD+CD272), memory (IgD2CD27+), and plasmablast (CD27++CD38+). Bars indicate mean 6 SD relative to condition without inhibitor. *p , 0.05, **p , 0.01, repeated measures one-way ANOVA followed by Dunnett multiple comparisons test (statistics were performed on nontransformed data). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Effect of kinase inhibitors on the function of healthy T cells. Healthy donor PBMC were stimulated with anti-CD3 and anti-CD28 Abs for 4 d in combination with JAK, BTK, and PI3Kd inhibitors (n =8).(A) Proliferation of CD4 and CD8 T cells after stimulation for 4 d. (B) Expression of activation marker CD25 on the surface of CD4 and CD8 T cells after 4 d of stimulation. (C) Levels of IFN-g in culture supernatants measured by ELISA. (D) Normalized values of (C), showing a clear relative effect of most kinase inhibitors on IFN-g production of T cells. (E) IFN-g production of MACS-purified CD4 and CD8 T cell fractions after 4 d of stimulation (n =4).(F) T cell subset differentiation after 4 d of stimulation. T cell subsets are defined as follows: naive (CD45RA+CD27+), memory (CD45RA2CD27+), effector (CD45RA2CD272), and RA-expressing effector memory (EMRA) (CD45RA+CD272). Bars indicate mean 6 SD relative to condition without inhibitor, except for (C), which depicts nontransformed data. *p , 0.05, **p , 0.01, repeated measures one-way ANOVA followed by Dunnett multiple comparisons test (statistics were performed on nontransformed data). these compounds, a thorough understanding of the effects of JAK, results indicate that JAK inhibitors do not have detrimental effects on BTK, and PI3Kd inhibitors on the function of both healthy and the function of healthy B cells (in contrast to BTK and PI3Kd in- malignant lymphocytes is warranted. JAK inhibitors could play a hibitors), the observed inhibition of NK and T cell function could beneficial role for the treatment of CLL by blocking signaling of have significant side effects during patient treatment. Off-target ef- important prosurvival molecules like IL-4 and IL-21. Although our fects on T cell function also occurs with ibrutinib, leading to 8 DIFFERENTIAL EFFECTS OF KINASE INHIBITORS ON LYMPHOCYTES Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Kinase inhibitors modulate allogeneic T cell responses. Healthy donor PBMC were stimulated with allogeneic LPS-maturated moDCs for 4 d in combination with JAK, BTK, and PI3Kd inhibitors (n = 8). (A) Proliferation of CD4 and CD8 T cells after coculture for 4 d. (B) Expression of activation marker CD25 on the surface of CD4 and CD8 T cells after 4 d of coculture. (C) Levels of IFN-g in culture supernatants measured by ELISA. Bars indicate mean 6 SD relative to condition without inhibitor. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, repeated measures one-way ANOVA followed by Dunnett multiple comparisons test (statistics were performed on nontransformed data). functional impairments, but these effects are not observed with the the context of CXCR4 signaling (38). If the effects of momelotinib more selective BTK inhibitor tirabrutinib. on BCR signaling are via on-target JAK2 inhibition, it is unclear We studied the effect of kinase inhibitors on healthy and ma- why ruxolitinib does not induce similar effects. Therefore, differ- lignant B cell function. Although momelotinib and ruxolitinib are ential off-target effects of momelotinib and ruxolitinib might ex- both JAK1/2 inhibitors, we observe clear differential effects on plain our observations. Momelotinib also inhibits TANK-binding BCR signaling. In particular, momelotinib inhibits BCR-mediated kinase 1 (TBK1) and IKKε, kinases that are involved in NF-kB phosphorylation of Akt and S6, whereas ruxolitinib did not target signaling upon activation by TLR and RIG1 signaling, suggesting BCR signaling. The inhibition of BCR signaling by momelotinib that these kinases play a role in the inhibitory effect of momelotinib might be beneficial during CLL therapy, as the BCR pathway plays on BCR signaling (39). an important role in the pathology of the disease (11). Recently, a Off-target effects of kinase inhibitors have been described [e.g., role for JAK2 has been described in BTK activation, especially in the observed effect of ibrutinib on T cell function is in line with The Journal of Immunology 9 reports on off-target ITK inhibition (26, 27)]. More specific BTK of T and NK cell function, and, consequently, the increased risk inhibitors, such as tirabrutinib, have been developed to limit off- of infections, may complicate clinical treatment with JAK inhib- target effects (33, 34). Tirabrutinib showed similar inhibition of itors in CLL patients. The BTK inhibitor tirabrutinib consistently B cell function in both healthy B cells and CLL cells but no in- showed a similar inhibitory potential as ibrutinib but lacked the off- hibition of T cell function. Allogeneic T cell responses were also target effects of ibrutinib on T cells, which warrants future research inhibited by ibrutinib but not tirabrutinib. Surprisingly, IFN-g comparing tirabrutinib with ibrutinib in the clinical setting. Con- levels increased in mixed lymphocyte reactions with both inhibi- versely, the inhibition of Tand NK cell function we observe by JAK tors, suggesting a BTK-mediated effect that is perhaps explained inhibitors and ibrutinib can be beneficial in disease settings with by BTK-dependent improvement of the Ag-presenting capacity of unwanted lymphocyte activation, such as GvHD. Our data are in dendritic cells (40). The disappearance of this effect with a higher line with early clinical data and demonstrate that kinase inhibitors dose of ibrutinib may well reflect ITK inhibition. in development as antitumor drugs in hematological malignancies Tirabrutinib may, therefore, have less infectious complications can also be applied to block unwanted lymphocyte function in other compared with ibrutinib. Together with promising phase 1 trial results diseases. The properties of individual kinase inhibitors can be (33, 34), these data support further exploration of tirabrutinib or exploited in combination treatment strategies tailored to the in- other more selective BTK inhibitors as a clinical treatment in CLL hibitory effects desired. and other malignancies. In contrast, off-target effects of ibrutinib on T cells are not always detrimental, as has been observed in CLL, in Disclosures which ibrutinib treatment increases T cell numbers and improves the S.T. was an employee of Gilead Sciences during this investigation. This re- efficacy of chimeric AgR T cell therapy (27, 41). search was sponsored via a research agreement between Amsterdam UMC Downloaded from Several prosurvival stimuli that CLL cells receive from the and Gilead Sciences. microenvironment are mediated via JAK signaling, like T cell– derived IL-4 and IL-21 (11, 12). In CLL patients, ruxolitinib leads to lymphocyte redistribution out of the lymph nodes and disease References stabilization, establishing in vivo activity of JAK inhibition (20, 1. Kipps, T. J., F. K. Stevenson, C. J. Wu, C. M. Croce, G. Packham, W. G. Wierda, 21). Although the efficacy of ruxolitinib monotherapy in patients S. O’Brien, J. Gribben, and K. Rai. 2017. Chronic lymphocytic leukaemia. Nat. Rev. Dis. Primers 3: 16096. http://www.jimmunol.org/ unfit for regular chemo-immunotherapy is only moderate, the 2. Byrd, J. C., J. R. Brown, S. O’Brien, J. C. Barrientos, N. E. Kay, N. M. Reddy, egress of tumor cells from lymph nodes observed with ruxolitinib S. Coutre, C. S. Tam, S. P. Mulligan, U. Jaeger, et al; RESONATE Investigators. treatment is similar to the lymphocytosis seen during the early 2014. Ibrutinib versus in previously treated chronic lymphoid leukemia. N. Engl. J. Med. 371: 213–223. phases of ibrutinib or idelalisib treatment (20, 21). JAK inhibi- 3. Roberts,A.W.,M.S.Davids,J.M.Pagel,B.S.Kahl,S.D.Puvvada,J.F.Gerecitano, tors may therefore be useful compounds in combination with T. J. Kipps, M. A. Anderson, J. R. Brown, L. Gressick, et al. 2016. Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia. N. Engl. J. Med. cell death–inducing agents, such as the Bcl-2 inhibitor venetoclax, 374: 311–322. even in patients refractory to ibrutinib or idelalisib, by depriving 4. Woyach, J. A., R. R. Furman, T. M. Liu, H. G. Ozer, M. Zapatka, A. S. Ruppert, tumor cells of microenvironmental stimuli. Because combination L. Xue, D. H. Li, S. M. Steggerda, M. Versele, et al. 2014. Resistance mecha- nisms for the Bruton’s tyrosine kinase inhibitor ibrutinib. N. Engl. J. Med. 370: treatment with venetoclax and ibrutinib has shown promising clinical

2286–2294. by guest on October 1, 2021 efficacy in CLL (42, 43), combinatory treatment of JAK inhibitors 5. Woyach, J. A., and A. J. Johnson. 2015. Targeted therapies in CLL: mechanisms and venetoclax may pose similar clinical benefit. of resistance and strategies for management. Blood 126: 471–477. 6. Herling, C. D., N. Abedpour, J. Weiss, A. Schmitt, R. D. Jachimowicz, The strong inhibitory effects on T and NK cell function by JAK O. Merkel, M. Cartolano, S. Oberbeck, P. Mayer, V. Berg, et al. 2018. Clonal inhibitors observed by us and others (23, 24) may complicate their dynamics towards the development of venetoclax resistance in chronic lym- phocytic leukemia. Nat. Commun. 9: 727. use in CLL. Ruxolitinib treatment is associated with an increased 7. Blombery, P., M. A. Anderson, J. N. Gong, R. Thijssen, R. W. Birkinshaw, risk of infectious complications, predominantly with infections of E. R. Thompson, C. E. Teh, T. Nguyen, Z. Xu, C. Flensburg, et al. 2019. Ac- the urogenital and respiratory tract, but reactivation of hepatitis B quisition of the recurrent Gly101Val mutation in BCL2 confers resistance to venetoclax in patients with progressive chronic lymphocytic leukemia. Cancer and tuberculosis also occurs (44). The increased risk of infections Discov. 9: 342–353. may pose a significant problem for already frail CLL patients, 8. Forconi, F., and P. Moss. 2015. Perturbation of the normal immune system in where infections are a leading cause of death (1). Indeed, rux- patients with CLL. Blood 126: 573–581. 9. Hamblin, A. D., and T. J. Hamblin. 2008. The immunodeficiency of chronic olitinib treatment led to infectious complications in unfit CLL lymphocytic leukaemia. Br. Med. Bull. 87: 49–62. patients (21). 10. van Attekum, M. H., E. Eldering, and A. P. Kater. 2017. Chronic lymphocytic leukemia cells are active participants in microenvironmental cross-talk. The functional impairment of lymphocytes by JAK inhibitors Haematologica 102: 1469–1476. can be beneficial in other disease settings, such as autoimmune 11. Aguilar-Hernandez, M. M., M. D. Blunt, R. Dobson, A. Yeomans, diseases and GvHD. Both T and B cells are implicated in GvHD S. Thirdborough, M. Larrayoz, L. D. Smith, A. Linley, J. C. Strefford, A. Davies, et al. 2016. IL-4 enhances expression and function of surface IgM in CLL cells. pathology (45), and ruxolitinib has clinical efficacy in refractory Blood 127: 3015–3025. GvHD patients (46). However, we show, in this study, that JAK 12. Pascutti, M. F., M. Jak, J. M. Tromp, I. A. Derks, E. B. Remmerswaal, inhibitors have relatively mild effects on B cell function, which R. Thijssen, M. H. van Attekum, G. G. van Bochove, D. M. Luijks, S. T. Pals, et al. 2013. IL-21 and CD40L signals from autologous T cells can induce may indicate that pathogenic B cell responses remain relatively antigen-independent proliferation of CLL cells. Blood 122: 3010–3019. intact in GvHD during ruxolitinib treatment. The inhibition of 13. Harrison, C., J. J. Kiladjian, H. K. Al-Ali, H. Gisslinger, R. Waltzman, V. Stalbovskaya, M. McQuitty, D. S. Hunter, R. Levy, L. Knoops, et al. 2012. both T and B cell function provides a rationale to use ibrutinib for JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. GvHD therapy. Indeed, our data demonstrate that ibrutinib also N. Engl. J. Med. 366: 787–798. inhibits T cells responses toward allogeneic cells. In clinical 14. Verstovsek, S., R. A. Mesa, J. Gotlib, R. S. Levy, V. Gupta, J. F. DiPersio, J. V. Catalano, M. Deininger, C. Miller, R. T. Silver, et al. 2012. A double-blind, trials, ibrutinib treatment led to clinical responses in two-thirds placebo-controlled trial of ruxolitinib for myelofibrosis. N.Engl.J.Med.366: 799–807. of refractory GvHD patients, with efficacy in all affected organs 15. Pardanani, A., R. R. Laborde, T. L. Lasho, C. Finke, K. Begna, A. Al-Kali, and a reduction in steroid use, and ibrutinib is now a Food and W. J. Hogan, M. R. Litzow, A. Leontovich, M. Kowalski, and A. Tefferi. 2013. Safety and efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis. Drug Administration–approved drug for glucocorticoid-resistant Leukemia 27: 1322–1327. GvHD (47). 16. Mesa, R. A., J. J. Kiladjian, J. V. Catalano, T. Devos, M. Egyed, A. Hellmann, D. McLornan, K. Shimoda, E. F. Winton, W. Deng, et al. 2017. SIMPLIFY-1: a In conclusion, we show that JAK inhibitors potently inhibit phase III randomized trial of momelotinib versus ruxolitinib in several prosurvival stimuli for CLL cells. However, their inhibition inhibitor-naı¨ve patients with myelofibrosis. J. Clin. Oncol. 35: 3844–3850. 10 DIFFERENTIAL EFFECTS OF KINASE INHIBITORS ON LYMPHOCYTES

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A 250K 250K 250K 250K 250K 250K 5 10 200K 200K 200K 200K 200K 200K

4 10 150K 150K 150K 150K 150K 150K CD19 FSC-H SSC-A SSC-A SSC-A SSC-A SSC-A 3 100K 100K 10 100K 100K 100K 100K

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300 e * e c c Ruxolitinib d 100 d 60 40 d e 40 20 200 d e i i v v Ibrutinib i i 40 50 % D 100 % D 20 20 Tirabrutinib 10 Idelalisib 20 0 0 0 0 0 1 2 3 4 0 1 2 3 4 - - 0 0 0 0 0 0 0 10 10 10 10 10 10 10 10 10 10 0 0 0 0 6 2 4 0 0 0 0 0 0 5 0 0 1 2 1 0 1 0 1 0 0 0 3 7 1 1 1 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10

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1 Supplemental Figure 1: Proliferation of CLL cells after treatment with kinase inhibitors 2 Proliferation of CLL PBMC was induced by co-culturing on 3T40L cells for 5 days while stimulating 3 with IL-21. (A) Gating strategy showing from left to right the gating of lymphocytes, viable cells, 4 and CD19+CD5+ CLL cells. (B) Representative histograms showing the proliferation of CLL PBMC 5 after 5 days in the unstimulated condition, the untreated condition, and the effects of kinase 6 inhibitors on proliferation. (C) Non-transformed proliferation data of CLL cells after CD40L+IL-21 7 stimulation and the influence of kinase inhibitors, corresponding to Figure 1A. Supplemental Figure 2 - Proliferation of healthy donor B cells after CpG and IL-2 stimulation.

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0 0 0 3 3 4 5 3 3 4 5 3 3 4 5 B C Unstimulated -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 Unstimulated 40 CpG + IL-2 100 100 CpG + IL-2 100 s 80 s 80 l l Momelotinib l l ** e 30 e ** ** * c 80 c 60 60 ** Ruxolitinib d d d e d e i 60 i Ibrutinib v v i 40 i 40 20 Tirabrutinib % D % D 4 4 4 40 20 20 10 10 10 Idelalisib 10 0 0 20 3 3 3 10 10 10 m 0 0 0 0 6 2 4 m 0 0 0 0 0 0 0 ti 5 0 0 1 2 ti 0 0 0 0 0 0 s 3 7 s 1 0 1 0 1 0 n n 1 1 1 0 0 U U 2 2 2 10 10 10 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 0 0 0 2 2 2 -10 -10 -10 CFSE CFSE 3 3 4 5 3 4 3 4 5 D -10 0 10 10 10 0 10 10 0 10 10 10 50 Unstimulated + 700 nM Mom + 24 nM Rux 2.0 1.2 50 CD40L + IL-21 s s 4 4 4 10 10 10 l l l 40 l 1.0 e e c 1.5 c Momelotinib 40 d d 0.8 3 3 3 10 10 10 d e d e Ruxolitinib i 30 * i v 1.0 v 0.6 i i * 30 ** * * Ibrutinib 2 2 2 10 10 10 % D % D 0 0 0

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0 0 0 1 Supplemental Figure 2: Proliferation of healthy donor B cells after CpG and IL-2 stimulation. 2 2 2 -10 -10 -10

3 3 4 5 3 4 3 4 5 -10 0 10 10 10 0 10 10 0 10 10 10 2 (A) Gating strategy for healthy B cells after CpG and IL-2 stimulation. From left to right, samples

4 4 4 10 10 10 3 were gated on lymphocytes, single cells, viable cells, and finally CD19+ B cells. (B) Representative 3 3 3 10 10 10

2 2 2 4 histograms of proliferation of healthy donor B cells after stimulation with CpG and IL-2 for 6 days, 10 10 10 0 0 0

2 2 2 -10 -10 -10

3 3 4 5 3 4 3 4 5 5 with and without kinase inhibitor treatment. (C) Non-transformed proliferation data of healthy B -10 0 10 10 10 0 10 10 0 10 10 10 6 cells after CpG and IL-2 stimulation, corresponding to Figure 2A (n=8). (D) Proliferation of healthy 7 B cells after stimulation with CD40L + IL-21 for 6 days, and the effect of kinase inhibitors (n=4). 8 (E) Viability of healthy B cells after 6 days of stimulation and treatment with kinase inhibitors. Bars

9 indicate mean ± SD. * = p < 0.05; ** = p < 0.01, repeated measures One-Way ANOVA followed

10 by Dunnett’s multiple comparisons test (statistics were performed on non-transformed data). Supplemental Figure 3 - Influence of kinase inhibitors on IL-2 + IL-12 + IL-18 stimulation of NK cells A B D Proliferation 200 150 IL-2 + IL-15 + 700 nM Mom + 24 nM Rux 1.5 1.2 1.2 60 150 * s s * l l l * l 100 1.0 * 1.0 *** s e e

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0.5 0.4 0.4 . 3 3 4 5 3 3 4 5 3 3 4 5 . 60 l l

-10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 % 100 e e R 100 0.2 R 0.2

120 + 1000 nM Ibru 30 + 1000 nM Tira + 1000 nM Idela 50 30 80 0.0 0.0 0.0 5 b b b b b 0 0 0 0 6 2 4 0 0 0 0 0 0 0 90 m 1 i i i i i im 5 0 0 1 2 im 0 0 0 0 0 0 60 i t 3 7 t 1 0 1 0 1 0 20 t - in in in in is s s 0 0 s IL t it t t l n n 1 1 1 n lo l u u la 3 4 5 3 4 5 U + e o r r e U U 0 10 10 10 0 10 10 10 60 x Ib b d 40 -2 m u a I L o ir I R M T M IL-2 + IL-12 + IL-18 100 10 M u u IL-2 + IL-12 + IL-18 M 1 M 30 20 M n 1 n 4 + u + 1 30 0 2 80 0 + + 0 0 0 7 3 3 4 5 3 3 4 5 3 3 4 5 + -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 60 E IFNγ production 20

1.2 1.4 40

* 10 ** * 1.2 1.0 ** ** 20 CFSE * + +

γ 1.0 γ 0.8 0 0 3 4 5 3 4 5 F N

F N 0 10 10 10 0 10 10 10 C I 0.8 0.6 % I %

.

. 0.6 l l e

e 200 4 4 4 0.4 80 10 10 10 R R 0.4 0.2 0.2 150 60 3 3 3 10 10 10 No drugs 0.0 0.0 100 0 0 0 0 6 2 4 0 0 0 0 0 0 0 40 im 5 0 0 1 2 im 0 0 0 0 0 0 2 2 2 t t 0 0 0 10 10 10 s 3 7 s 1 1 1 n n 1 1 1 0 0 0 U U 2 2 2 50 -10 -10 -10 20

3 3 4 5 3 4 3 4 5 -10 0 10 10 10 0 10 10 0 10 10 10 IL-2 + IL-12 + IL-18 IL-2 + IL-12 + IL-18 0 0 3 4 5 3 4 5 0 10 10 10 0 10 10 10

4 4 4 TNFα production 10 10 10 F 150

1.2 1.2 300 3 3 3 * 10 10 10 * ** + 700 nM Mom 1.0 ** ** 1.0 100 * *** 200 + + ** 2 2 2 α 10 10 10 α 0.8 *** 0.8 **** F 0 0 0 F

2 2 2 **** -10 -10 -10 50 0.6 0.6 100 % T N 3 3 4 5 3 4 3 4 5 % T N

-10 0 10 10 10 0 10 10 0 10 10 10 . . l l e e 0.4 0.4 R R 0 0 3 4 5 3 4 5 4 4 4 0 10 10 10 0 10 10 10 10 10 10 0.2 0.2

0.0 0.0 120

3 3 3 10 10 10 0 0 0 0 6 2 4 0 0 0 0 0 0 0 m 5 0 0 1 2 m 0 0 0 0 0 0 + 24 nM Rux ti 3 7 ti 1 0 1 0 1 0 s s 150 n n 1 1 1 90 U U 2 2 2 10 10 10

0 0 0 IL-2 + IL-12 + IL-18 IL-2 + IL-12 + IL-18 100 2 2 2 60 -10 -10 -10

3 3 4 5 3 4 3 4 5 -10 0 10 10 10 0 10 10 0 10 10 10 G Degranulation 50 30

CD56 4 4 4 10 10 10 1.2 1.2 0 0 3 4 5 3 4 5 * * 0 10 10 10 0 10 10 10 3 3 3 10 10 10 1.0 1.0 *** + + *** 200 a + 1000 nM Ibru a 7 7 80 0 0.8 0 0.8 1 2 2 2 1 10 10 10 150 D 0 0 0 0.6 D 0.6 2 2 2 60 -10 -10 -10 % C % C

. 3 3 4 5 3 4 3 4 5 . l -10 0 10 10 10 0 10 10 0 10 10 10 0.4 l 0.4 100 e e 40 R R 0.2 0.2 4 4 4 10 10 10 50 20 0.0 0.0

0 0 0 0 6 2 4 0 0 0 0 0 0 0 0 0 3 3 3 m m 10 10 10 ti 5 0 0 1 2 ti 0 0 0 0 0 0 s 3 7 s 1 0 1 0 1 0 3 4 5 3 4 5 n n 1 1 1 0 10 10 10 0 10 10 10 + 1000 nM Tira U U

2 2 2 10 10 10 IL-2 + IL-12 + IL-18 IL-2 + IL-12 + IL-18 0 0 0

2 2 2 -10 -10 -10

3 3 4 5 3 4 3 4 5 -10 0 10 10 10 0 10 10 0 10 10 10 H Cytotoxicity

4 4 4 Unstimulated 10 10 10 1.2 1.2 s s * * IL-2 + IL-12 + IL-18 i i * * ** s 3 3 3 s 1.0 1.0 * 10 10 10 *** y y *** l l **

+ 1000 nM Idela Momelotinib c c i i 0.8 0.8 f f i i 2 2 2 c 10 10 10 c Ruxolitinib 0 0 0 0.6 0.6 p e 2 2 2 p e -10 -10 -10 Ibrutinib % S 3 3 4 5 3 4 3 4 5 % S 0.4 0.4

-10 0 10 10 10 0 10 10 0 10 10 10 . . l l e e Tirabrutinib R IFNγ TNFα CD107a R 0.2 0.2 0.0 0.0 Idelalisib m 0 0 0 0 6 2 4 m 0 0 0 0 0 0 0 ti 5 0 0 1 2 ti 0 0 0 0 0 0 s 3 7 s 1 0 1 0 1 0 n n 1 1 1 U U IL-2 + IL-12 + IL-18 IL-2 + IL-12 + IL-18

1 Supplemental Figure 3: Influence of kinase inhibitors on IL-2 + IL-12 + IL-18 stimulation of NK cells 2 (A) Representative histograms of NK cell proliferation after stimulation with IL-2 and IL-15 for 5 days, and the influence 3 of kinase inhibitors. (B) Viability of NK cells after 5 days of IL-2 + IL-15 stimulation in the presence of kinase inhibitors. 4 (C) Gating strategy of IFNγ and TNFα producing, and degranulating (CD107a+) NK cells after stimulation with K562 5 cells. (D) Proliferation of NK cells after stimulation with IL-2 + IL-12 + IL-18 for 5 days (n=8). (E+F) Percentage of NK 6 cells producing IFNγ (E) or TNFα (F) after co-culture of IL-2 + IL-12 + IL-18 stimulated PBMC with K562 target cells 7 (n=8). (G) Percentage of degranulated (CD107a+) NK cells after co-culture with K562 target cells (n=8). (H) Specific 8 lysis of K562 target cells after co-culture with IL-2 + IL-12 + IL-18 stimulated PBMC for 3 hours (n=4). Bars indicate 9 mean ± SD relative to condition without inhibitor, * = p < 0.05; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, repeated

10 measures One-Way ANOVA followed by Dunnett’s multiple comparisons test (statistics were performed on non- 11 transformed data). Supplemental Figure 4 - Effect of kinase inhibitors on proliferation, activation and differentiation of T cells after anti-CD3 and anti-CD28 stimulation

A CD4 CD8 B CD4 CD8 D CD4

120 1.5 200 5 5 10 10

90 150 s l

4 4 l 10 10 e 1.0 c

100 60 αCD3 + αCD28 e 3 3 10 10 b l a i 50 30 0 0 V 0.5 % 3 3 -10 -10 0 0

3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 100 0.0 5 5 10 10 8 b b b b b 80 30 m i i i i i ti 2 n n n n s s D ti ti ti ti li n C o li u u a 4 4 α l r r l 10 10 U / e o e 60 3 x Ib b d m u ra I 20 D o R M i + 700 nM Mom C u T M α M M u n 1 M 1 40 3 3 M u 10 10 n 4 + + 0 2 1 10 0 + + 0 0 7 20 +

3 3 -10 -10 0 0 3 4 5 3 4 5 0 10 10 10 0 10 10 10 3 4 5 3 4 5 CD8 0 10 10 10 0 10 10 10 1.5 5 5 200 10 10 80

4 4 s 150 10 10 60 l l

e 1.0

+ 24 nM Rux c 100 40 3 3 e 10 10 b l a

50 20 0 0 i V 0.5 3 3

-10 -10 % 0 0

3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

150 5 5 CD4 10 CD8 10 0.0 300 8 b b b b b im 2 i i i i i t D in in in in is 4 4 s t it t t l 100 10 10 n C lo l u u la U /α e o r r e 200 3 x Ib b d m u a I + 1000 nM Ibru D o R M ir C M u T M 3 3 α u 10 10 M 1 M n M 1 50 + u 100 n 4 + 0 2 1 0 0 0 + + 7 + 3 3 -10 -10 0 0

3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

120

5 5 10 10

150 90

4 4 10 10

100 60 + 1000 nM Tira 3 3 10 10

50 30 0 0

3 3 -10 -10 0 0

3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

200 5 5 10 10 80

150 4 4 10 10 60

100 + 1000 nM Idela 40 3 3 10 10

50 20 0 0

3 3 -10 -10 0 0

3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

CFSE CD25 C Differentiation CD4 CD8 120 * 120 EMRA s s 100 100 l l Effector l l e e c c 80 80 Memory T T

8 4 60 60 Naïve D D C C

40 40 o f o f % % 20 20

0 0 0 0 im M M M M M M im M M M M M M t n n n n n n t n n n n n n s 0 0 0 6 2 4 s 0 0 0 6 2 4 n 5 0 0 1 2 n 5 0 0 1 2 U 3 7 x U 3 7 x m u x x m u x x o m m R u u o m m R u u M o o R R M o o R R M M M M

αCD3 + αCD28 αCD3 + αCD28

1 Supplemental Figure 4: Effect of kinase inhibitors on proliferation, activation and differentiation of T cells 2 after anti-CD3 and anti-CD28 stimulation 3 (A) Representative histograms of proliferation of healthy donor CD4 and CD8 T cells after anti-CD3 and 4 anti-CD28 stimulation for 4 days. (B) Activation of healthy donor CD4 and CD8 T cells after anti-CD3 and 5 anti-CD28 stimulation for 4 days. (C) Effect of momelotinib and ruxolitinib on T cell differentiation during 6 activation (n=8). (D) Viability of CD4 and CD8 T cells after 4 days of stimulation in the presence of kinase

7 inhibitors. Bars indicate mean ± SD relative to condition without inhibitor, * = p < 0.05, repeated measures

8 One-Way ANOVA followed by Dunnett’s multiple comparisons test.