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Mouse models
Flow cytometry and functional assays
Single cell suspensions from peripheral blood (PB) or lymphoid tissues were prepared and flow cytometric analyses of cell surface proteins and transcription factors were performed as detailed in the Online Supplementary Appendix and described before.8,27 Gating strategies are depicted in the Online Supplementary Appendix and antibodies are listed in Online Supplementary Table S1.
Cytokine release, granzyme B production and degranulation capacity of CD8+ T cells were assayed as previously described with minor modifications, as outlined in the Online Supplementary Appendix.9
Human PB mononuclear cells (PBMC) or mouse splenocytes were pre-treated for 30 minutes (min) with ibrutinib, CC-292 or ACP-196 (Selleckchem, Munich, Germany), then stimulated with 1 mg/mL αCD3 antibodies (clone HIT3a or 145-2C11, respective- ly) in 96-well round-bottom microtiter plates at a density of 2x106/mL and incubated at 37°C and 5% CO2 until analyzed by flow cytometry. For assessment of proliferation, cells were stained with 5 mM carboxyfluoresceinsuccinimidyl ester (CFSE; eBioscience) prior to drug treatment and stimulation as previously described.28
Statistical analysis
Sample size was determined based on expected variance of read-out. No samples or animals were excluded from the analyses. No randomization or blinding was used in animal studies. Data were analyzed using Prism 5.04 GraphPad software. Statistical tests of data were one-way ANOVA followed by multiple com- parison test or unpaired Student t-test with Welch approximation to account for unequal variances. When appropriate, paired Student t-test was used. P<0.05 was considered to be statistically significant. All graphs show means±standard error of mean, unless otherwise indicated.
Results
Ibrutinib modulates effector CD8+ T-cell differentiation and proliferation in the TCL1 adoptive transfer model
To investigate effects of ibrutinib on CD8+ T cells in vivo,
Em-TCL1 (TCL1) mice on C57BL/6 background were kindly provided by Dr. Carlo Croce (Ohio State University, OH, USA).23 C57BL/6 wild-type (WT) mice were purchased from Charles River Laboratories (Sulzfeld, Germany), and Nr4a1GFP mice24 were kindly provided by Dr. Markus Feuerer (DKFZ, Heidelberg, Germany).
haematologica | 2021; 106(4)
Combining ibrutinib and checkpoint blockade in CLL
CLL cells.13 Gunderson et al. have demonstrated that BTK inhibition in tumor-associated myeloid cells reprograms them towards M1-like immunostimulatory phenotypes resulting in enhanced anti-tumoral T-cell activity.14 In addi- tion to modulating BTK-expressing cells in the microenvi- ronment, ibrutinib has been shown to impact natural killer (NK)- and T-cell activity due to its non-BTK effects on IL- 2-inducible T-cell kinase (ITK).15,16 In a preclinical study, ITK inhibition by ibrutinib was shown to impair Fc recep- tor-mediated NK-cell functions and to antagonize cytotox- icity of rituximab.16 However, NK-cell-mediated cellular cytotoxicity was significantly recovered 12 hours (h) after ibrutinib has been removed, which may be attributed to turnover of ITK.17 Inhibition of ITK by ibrutinib was fur- ther shown to modulate TCR signaling in CD4+ T cells and enhance Th1 response in vitro.15 In addition, a reduced num- ber or percentage of regulatory T cells in peripheral blood of ibrutinib-treated patients was reported.18,19 Interestingly, ibrutinib was recently approved for the treatment of refrac- tory chronic graft-versus-host-disease (cGvHD) after allo- geneic hematopoietic stem cell transplantation, indicating its potent immunomodulatory effect in vivo.20
The strong immunomodulatory activity of ibrutinib encouraged several pre-clinical and clinical studies assess- ing its potential combination with other immunotherapeu- tic drugs, primarily immune checkpoint blockade.21 The success of these immunotherapeutic approaches relies on enhancing the functional capacities and proliferation of anti-tumoral CD8+ T cells. Despite a large body of evidence highlighting the immunomodulatory activity of ibrutinib, its exact impact on CD8+ T cells in CLL remains less defined. While Long et al. observed an increase in CD4+ and CD8+ T-cell numbers in ibrutinib-treated patients,19 a drop in T-cell numbers, activation, and proliferation was reported in another patient cohort.22 Moreover, it was sug- gested that ibrutinib can enhance anti-tumoral CD8+ T-cell function in CLL patients by fostering a switch of T-helper- cell polarization towards anti-tumoral Th1 cells.15 However, this change in Th1 polarization could not be confirmed in a cohort of ibrutinib-treated patients.19 Thus, in this study, we utilized the Em-TCL1 adoptive transfer (TCL1 AT) model of CLL to monitor CD8+ T-cell differen- tiation and function under ibrutinib treatment. Through this, we observed that this drug reduces the functionality of CD8+ T cells which could be overcome by combining ibrutinib with immune checkpoint blockade in the TCL1 AT model. The relevance of these findings for therapeutic applications has to be proven in ongoing clinical trials com- bining ibrutinib with anti(α)PD-L1 antibody durvalumab for treatment of B-cell lymphoma and CLL patients. (Trial registered at clinicaltrials.gov identifiers: NCT02401048 and NCT02733042).
Methods
TCL1 tumor cells of C57BL/6N background were propagated once by adoptive transfer in C57BL/6N mice to ensure having
enough tumor cells from the same donor for all treatment arms. Adoptive transfer of TCL1 tumors was performed as previously described.9,25 Briefly, 1-2x107 leukemic TCL1 splenocytes (CD5+CD19+ content of purified cells was typically above 90%) were transplanted by intraperitoneal (i.p.) injection into 2-3- month-old C57BL/6N WT females. All animal experiments were carried out according to governmental and institutional guidelines and approved by the local authorities (Regierungspräsidium Karlsruhe, permit numbers: G-36/14, G-123/14, G-16/15, and G- 53/15).
In vivo treatment
Two to three weeks after tumor cell transplantation, tumor load
in blood (defined as the number of CD5+CD19+ CLL cells/mL) was measured, and mice were assigned to different treatment arms to achieve comparable tumor load in all groups at baseline prior to treatment. Ibrutinib (provided by Pharmacyclics LLC, an AbbVie Company) was administered in drinking water containing sterile control vehicle (1% HP-β-CD) at a concentration of 0.16 mg/mL, as previously described.26 For PD-1/PD-L1 blockade, mice were injected i.p. with 0.2 mg of αPD-1 (clone: RMP1-14), αPD-L1 (clone: 10F.9G2), or rat IgG2a isotype control antibody (clone: 2A3; all from BioXcell, West Lebanon, NH, USA) every 3 days for 4 weeks.
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