*2.3. Proliferation Analysis by 3H-Thymidine Incorporation*

Cell proliferation was analyzed by 3H-thymidine incorporation assay. For T cell activation, PHA was added to PBMC suspension and 1 × <sup>10</sup><sup>5</sup> PBMCs/well were seeded into flat bottom 96-well plates. Different concentration (1 nM–10 μM, 1:10 serial dilution) of the four investigated JAKi were added as triplicates either directly to the cell culture or 48 h after PHA-stimulation (preactivated lymphocytes). After 72 h of PHA activation, PBMCs were pulsed with [3H]-thymidine at a dose of 0.2 μCi/well for additional 6 h. At the end of the incubation period cells were harvested and 3H-thymidine incorporation was quantified using the microplate liquid scintillation counter Wallac MicroBeta TriLux from Perkin Elmer (Waltham, MA, USA).

#### *2.4. Proliferation Analysis by the CFSE Dilution Assay*

Additionally, cell proliferation was assessed by the cell trace carboxyfluorescein succinimidyl ester (CFSE) cell proliferation kit (Invitrogen, Carlsbad, CA, USA). Therefore, freshly isolated PBMCs were washed in phosphate buffers saline (PBS; PAN-Biotech) and resuspended in 1 mL PBS containing 5% inactivated fetal calf serum (FCS). Subsequently, 5 μM CFSE solution was added and incubated for 5 min at room temperature in the dark. Afterwards, cells were washed twice in PBS-FCS and were resuspended to a final concentration of 1 × <sup>10</sup><sup>6</sup> PBMC/mL in AIM-V medium. CFSE loaded unstimulated cells served as control sample, representing CFSEhigh, non-divided cell population. For activation PHA was added to remaining CFSE-stained PBMC suspension, which was subsequently plated into 24-well cell culture plates. Increasing concentrations of JAKi or MTX were either added directly or 48 h after PHA-stimulation. After an incubation period of 96 h PBMCs were transferred into 5 mL round bottom polystyrene tubes and washed once in cold PBS containing 0.5% BSA. Subsequently, CFSE intensity of living cells (gating based on forward/side scatter signal) was determined in FITC channel by flow cytometry (LSRFortessa cell analyzer, BD Biosciences, Mountain View, CA, USA) and FlowJo software (version 7.6.4, Tree Star Inc., Ashland, OR, USA).

### *2.5. Analysis of CD25 Expression*

Activation status was assessed by CD25 expression. Therefore, 1 × <sup>10</sup><sup>6</sup> PBMCs/sample were simultaneously treated for 48 h with PHA and different JAKi at various concentrations as indicated. Afterwards, cells were transferred into 5 mL round bottom polystyrene tubes, washed twice with PBS containing 0.5% bovine serum albumin (BSA; AppliChem, Darmstadt, Germany) and incubated for 30 min at 4 ◦C with 1:200 diluted phycoerythrin(PE)-coupled anti-human CD25 antibody (BioLegend, San Diego, CA, USA). Subsequently, samples were washed with PBS containing 2 mM ethylenediaminetetraacetic acid (EDTA) and resuspended in PBS-BSA. Samples were kept cold until flow cytometry analysis using a BD LSRFortessa cell analyzer. Data were acquired by FACSDiva 6.0 software (BD Biosciences) and analyzed by FlowJo software.

#### *2.6. Viability Assessment*

JAKi- and MTX- induced cell death was determined after 72 h in unstimulated, freshly PHA-stimulated and preactivated PBMCs using a FITC Annexin V/propidium iodide (PI) apoptosis detection kit (BioLegend, San Diego, CA, USA). In brief, 1 × 106 PBMC were transferred into 5 mL round bottom polystyrene tubes, washed once in cold PBS-BSA and resuspended in 50 μL staining solution, comprising Annexin V and PI diluted 1:20 in Annexin V binding buffer. After 15 min incubation at room temperature in the dark cellular staining was terminated by addition of 200 μL binding buffer. Ratio of viable (Annexin V−/PI<sup>−</sup>), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+), and necrotic (Annexin V−/PI+) cells was determined of 20,000 cells/sample by BD LSRFortessa cell analyzer and subsequent analysis utilizing FlowJo software. Unstained, single- and doublestained cells treated with camptothecin were included as control samples.

#### *2.7. Detection of γH2AX and 53BP1 Foci*

Automated quantification of intranuclear γH2AX and 53BP1 foci, described as sensitive indicators for DNA double-strand breaks (DSB), was performed to study JAKi-induced DNA DSB and their impact on DNA repair of radiation-induced DSBs [25–27]. Therefore, unstimulated PBMCs were treated with indicated concentrations of JAKi or MTX. Additionally, one fraction was exposed to γ-rays at a dose of 2 Gy (Biobeam 8000, Cs 137, Gamma-Service Medical, Leipzig, Germany) to induce DNA damage. After an incubation period of 24 h non-irradiated and radiated samples were harvested, washed in PBS and fixed for 15 min with 1% formaldehyde on silanized glass slides, as described in detail elsewhere [28,29]. Subsequently, cells were permeabilized in 0.2% Triton X-100, washed in blocking buffer (PBS containing 1% BSA) and incubated for 60 min at room temperature simultaneously with 1:1000 diluted γH2AX (anti-phosphohistone H2AX mouse monoclonal IgG primary antibody, clone JBW301, Millipore, Schwalbach, Germany) and 53BP1 primary antibodies (anti-53BP1 rabbit polyclonal IgG (NB 100–305), Novus Biologicals, Centennial, CO, USA). Afterwards, slides were washed and incubated for 1 h at room temperature with 1:500 diluted polyclonal goat anti-mouse IgG antibody conjugated to Alexa Fluor 488 and polyclonal goat anti-rabbit IgG antibody conjugated to Alexa Fluor 647 (Lifetechnologies, Darmstadt, Germany). After a final washing cycle in PBS, slides were covered with DAPI (4 ,6-diamidino-2-phenylindole)-containing mounting medium (Medipan, Berlin/Dahlewitz, Germany). Directly after staining procedure slides were analyzed by an automated digital microscopy system (AKLIDES, Medipan, Berlin/Dahlewitz, Germany) quantifying the number of γH2AX and 53BP1 foci in 300 nuclei per sample [28,29].

#### *2.8. Statistical Analysis*

Quantitative data analysis was performed by GraphPad Prism software version 5.01 (Graph Pad Software, La Jolla, CA, USA). Half-maximal inhibitory dose (IC50) was calculated by non-linear regression from logarithm-transformed data. Significance levels among samples treated with the same JAKi or MTX were calculated by repeated measures ANOVA

(analysis of variance) with 95% confidence interval (α = 0.05) followed by the Dunnett's post-hoc test, to compare the results with DMSO-treated control group. Data in text and figures are displayed as the mean ± standard error of the mean (SEM), and *p* values are indicated by asterisks (\*\*\* *p* ≤ 0.001; \*\* *p* ≤ 0.01; \* *p* ≤ 0.05).

#### **3. Results**

#### *3.1. Impact of JAKi and MTX on Lymphocyte Activation and Proliferation*
