Supplementary Materials for
Microenvironmental regulation of the IL-23R/IL-23 axis overrides
chronic lymphocytic leukemia indolence
Giovanna Cutrona,* Claudio Tripodo, Serena Matis, Anna Grazia Recchia, Carlotta Massucco, Marina Fabbi, Monica Colombo, Laura Emionite, Sabina Sangaletti,
Alessandro Gulino, Daniele Reverberi, Rosanna Massara, Simona Boccardo, Daniela de Totero, Sandra Salvi, Michele Cilli, Mariavaleria Pellicanò, Martina Manzoni,
Sonia Fabris, Irma Airoldi, Francesca Valdora, Silvano Ferrini, Massimo Gentile, Ernesto Vigna, Sabrina Bossio, Laura De Stefano, Angela Palummo, Giovanni Iaquinta,
Martina Cardillo, Simonetta Zupo, Giannamaria Cerruti, Adalberto Ibatici, Antonino Neri, Franco Fais, Manlio Ferrarini, Fortunato Morabito
*Corresponding author. Email: [email protected] Published 14 February 2018, Sci. Transl. Med. 10, eaal1571 (2018)
DOI: 10.1126/scitranslmed.aal1571
The PDF file includes: Materials and Methods
Fig. S1. Gating strategy to analyze IL-23R complex induction after activation by CD40L-expressing fibroblasts.
Fig. S2. IL-12Rβ1 mRNA production after CLL cell activation by CD40L-expressing fibroblasts.
Fig. S3. Expression of a complete 23R by CLL cells after depletion of IL-23R–positive cells and coculture with autologous activated T cells.
Fig. S4. Demonstration of intracytoplasmic IL-23 chains in CLL cells. Fig. S5. HS5 stromal cells do not express CD40L.
Fig. S6. Enhanced IL-23R complex expression by coculture of PBMC with NLC. Fig. S7. IL-23R complex expression by CLL cells cocultured with stromal cells or NLCs.
Fig. S8. Relationship between the BCR signaling pathway and IL-23R complex expression in CLL cells.
Fig. S9. Silencing of IL-23A or IL-23R genes by siRNA.
Fig. S10. STAT3 phosphorylation induced by CLL cell exposure to IL-23. Fig. S11. NSG mice engrafted with CLL cells.
Fig. S12. Minimal residual disease detection in mice tissues after treatment with αIL-23p19.
Fig. S13. Anti–IL-23p19 mAb does not induce antibody-dependent cytotoxicity. Fig. S14. Schematic representation of the IL-23R complex/IL-23 axis in CLL. Table S1. Relationship between IL-23R chain expression and prognostic parameters.
Table S2. Univariate and multivariate bootstrapping validated Cox regression analysis of TTFT.
Table S3. Features of the CLL cases used in coculture experiments with CD40L-expressing fibroblasts (CD40L-TC).
Table S4. Summary of the features of all CLL cases whose cells were used for xenograft tests.
References (50–53)
Other Supplementary Material for this manuscript includes the following: (available at
www.sciencetranslationalmedicine.org/cgi/content/full/10/428/eaal1571/DC1) Table S5 (Microsoft Excel format). Primary data.
Materials and Methods
CLL Patients, CLL cell preparation and prognostic marker determination
Newly diagnosed CLL patients from participating Institutions were enrolled within 12 months from diagnosis (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540). Diagnosis was confirmed by flow cytometry analysis together with the determination of CD38 and ZAP-70 expression, IGHV mutational status and of cytogenetic abnormalities as previously described (28). TP53 mutational status was determined by RT-PCR as previously described (35).
PBMCs from patients with CLL were isolated by Ficoll-Hypaque (Seromed, Biochrom) density gradient centrifugation. In selected experiments CD19-positive CLL cells were enriched by negative selection with the EasySep-Human B-cell Enrichment Kit without CD43 depletion (STEMCELL Technologies, Voden Medical Instruments S.p.A.). The percentage of purified B cells (CD19+) exceeded 95%.
Flow cytometry
IL-23R complex was detected by quadruple staining with CD19 PE-CY7 (BD Biosciences, Inc, San Jose, CA), CD5 APC (BD Biosciences), anti-human IL-23R PE (R&D Systems Inc., Minneapolis, MN, USA), anti-human IL-12Rβ1 FITC (R&D Systems). The cells were analyzed using a FACSCanto flow-cytometer and DIVA 6 (BD Biosciences) or FLOWJO V.9.8.3 software (Treestar Inc.). For data analysis CD5/CD19-double positive cells were gated within the SSC/FSC lymphocyte gate. The percentage of IL-23R and IL-12Rβ1 was analyzed on the gated population.
CD80 and CD86 expression was determined by single staining with CD80 PE or CD86 PE (BD Biosciences).
Association of the IL-23R complex with CD38 activation marker was performed by quadruple staining with CD19 PE-CY7, CD38 APC (BD Biosciences), 23R PE, and IL-12R1 FITC mAbs (R&D Systems). Association of the IL-23R complex with the Ki67 proliferation marker was evaluated by surface staining with CD19 APC-H7, CD5 PerCP-CY5.5 (BD Biosciences), IL-23R PE, and IL-12R1 APC (R&D Systems), followed by cell fixation and permeabilization (Fix & Perm Invitrogen, ThermoFisher Scientific) and staining for Ki67 FITC (clone MIB-1, DakoCytomation S.r.L).
CLL cell co-culture
For in vitro activation with CD40L, enriched CLL cells were cultured in the presence of a stable CD40L-expressing 3T3 (CD40L-TC) murine fibroblast cell line or a control NIH-3T3 cell line stably transfected with the pIRES vector alone (Mock), -irradiated to avoid cell proliferation (29).
For CLL activation by autologous T-cell, CLL PBMCs were cultured in RPMI medium in the presence of Dynabeads Human T-activator CD3/CD28 (Gibco, ThermoFisher Scientific; beads to T cells ratio 1:1) and of recombinant IL-2 (Proleukin, Norvartis, 30 U/mL), which was added on alternate days. Cell cultures were monitored daily until substantial clumping indicating cell activation was observed (up to 360 h), then expression for IL-12Rβ1 and IL-23R chains of the IL-23R complex was performed by flow cytometry
The effects of IL-23 or of αIL-23p19 neutralizing antibody on the induction IL-23R complex by activated T cells activation was evaluated as follows: PBMCs from CLL patients were stimulated with CD3/CD28 beads and IL-2 for 120-h, subsequently CLL were purified by negative selection as described above, and exposed to IL-23 100 ng/mL (rIL-23, R&D, Systems) or to anti-Human IL-23p19 Functional Grade Purified neutralizing IgG1 kappa monoclonal
antibody (clone HNU2319, eBioscience) 10 μg/mL, or a mix of both, for an additional 48 h. Cell viability was determined by AnnexinV and PI staining and flow. Kinase inhibitors ibrutinib (BTK inhibitor, PCI-32765, Selleckchem), tofaticinib (Jak3/1/2 inhibitor, CP-690550 MedChem Express, and ruxolitinib (Jak1/2 inhibitor, INCB018424, MedChem Express) were diluted in DMSO and tested at concentrations of 0.5, 1, and 5 μM. For in vitro experiments the concentration of 1 μM was found to be the most appropriate for each of the inhibitors tested.
The expression of the complete IL-23R complex or components thereof was evaluated by flow cytometry before and after co-culture of purified CLL cells with the HS5 stromal cell line (31) or with autologous nurse-like cells (NLC) (32). Briefly 1×106 CLL cells/well were
incubated with or without 15×103 HS5/well for 72 h. Subsequently CLL cells were harvested and
cell viability was evaluated by DiOC6 (3,3′-dihexylocarbocyanine iodide) (Sigma-Aldrich) staining, as previously described (50). Isolation of nurse-like cells together with the co-culture conditions of these cells with CLL cells have previously been reported in detail (32). Briefly 105
purified monocytes/well were cultured with 2×106 autologous CLL cells for 14 days.
Subsequently CLL cells in suspension were washed out and 2×106 CLL cells from a new batch
of cryopreserved cells from the same patient were added to the culture, where typical NLC were adherent to the plate. The same concentration of CLL cells was further cultured in only medium for controls. After 7 days of culture CLL cells were harvested and cell viability was evaluated by DiOC6. In these experiments CLL cells were purified by negative selection with the EasySep-Human B-cell Enrichment Kit without CD43 depletion (STEMCELL Technologies).
CD40L and CD105 expression were determined by single staining with CD40L PE (BD Biosciences) or CD105 FITC (Immunotools).
To assess their capacity of being stimulated via BCR signaling, CLL cells. CLL cells were cultured with Dynabeads M-450 Epoxy (Invitrogen, ThermoFisher Scientific) coated with
(10 μg/1×107 beads) Goat anti-Human IgM μ chain specific (Gαμ-Ab, Southern Biotechnology,
Birmingham, AL) and/or IL-4 25 ng/mL (Gibco, Thermo Fisher Scientific) as described (33).
IL-23 detection
IL-23 production in culture supernatants was measured by Human Cytokine/Chemokine Panel II and Luminex® MAGPIX System® (Merck Millipore)
Protein phosphorylation detection and measurement
Purified CLL cells (5×106) were exposed to IL-23 (200 ng) in 250 L of RPMI medium
at 37°C for 10, 30 or 60 minutes or, in a separate set of experiments CLL cells were exposed to 10 μg of anti-μ-Ab (G-Ab; Southern Biotechnology), Goat anti human IgD anti--Ab (G-Ab; Southern Biotechnology), or a combination of both, at 37°C for 5 minutes.
Cells exposed to appropriate stimuli were centrifuged and cell pellets lysed with 100 μL of buffer containing 2% Triton-X-100, 20 mM Tris HCl pH 8, 2 mM EDTA, 2x Protease Inhibitors (Complete C Mini, Roche, Sigma-Aldrich), and 2 μM sodium orthovanadate in saline solution. Nuclei-free cell extracts from 106 cells were resolved by sodium dodecylsulfate
electrophoresis on 10% polyacrylamide gels under reducing conditions, and then transferred onto nitrocellulose membranes (Hybond C Extra; GE Healthcare Life Sciences, GE Healthcare Europe GmbH). Membranes were incubated with the primary antibodies followed by goat anti-mouse or goat anti-rabbit (DakoCytomation) peroxidase-labeled secondary antibodies. Peroxidase activity was detected by chemiluminescence (ECL-PRIME, GE Healthcare Life Sciences). Protein bands were analyzed using ImageJ Analysis Software version 1.48. Band data were generated by analyzing unsaturated autoradiographic images or by the Western blot imaging system Alliance Mini HD9 (Uvitec).
Membranes were incubated with the following antibodies: anti-phospho-STAT3 (pSTAT3) (Tyr705) mAb clone 4/pSTAT3, anti-total STAT3 (tSTAT3) mAb 84/STAT3 (BD Transduction Laboratories, Biosciences), rabbit anti-phosphorylated-BTK (pBTK) (p-Y223) (Abcam, Cambridge), anti-total BTK mAb clone 566928 (R&D Systems), and anti-tubulin-alpha mAb clone B-5-1-2 (Sigma-Aldrich) in 0.5% BSA-TBST, followed by goat anti-mouse- or goat anti-rabbit (DakoCytomation) peroxidase-labeled secondary antibodies and chemiluminescence (ECL-PRIME, GE Healthcare Life Sciences).
To determine the percentage of pSTAT3 or pBTK positive CLL cells, by flow cytometry, cells were stained with CD19 APC and then fixed (BD Cytofix Buffer, BD Phosflow, BD Biosciences), permeabilized (BD Phosflow perm Buffer III, BD Biosciences), stained with pSTAT3 (Tyr705) rabbit mAb Alexa Fluor 488 conjugate (clone D3A7, Cell Signaling Technology) or pSTAT3 (Tyr705) mouse mAb APC conjugate (clone LUVNKLA, ThermoFisher Scientific) or pBTK Alexa Fluor 488 conjugate (clone N35-86, BD Phosflow, BD Biosciences), and analyzed by flow cytometry.
In situ studies
For immunohistochemistry (IHC) and immunofluorescence (IF) analyses on human tissue specimens, LN samples from 16 CLL patients were selected from the archives of the Department of Health Sciences, Human Pathology Area, University of Palermo. Single-marker analysis was performed on four-micrometer-thick sections from formalin-fixed and paraffin-embedded specimens as previously described (51). Briefly sections were deparaffinized through graded alcohol series and rehydrated in water prior to heat-mediated antigenic retrieval in a thermostatic bath. The antigen unmasking technique was performed using Novocastra Epitope Retrieval Solutions pH 6 and pH 9 in PT Link (DakoCytomation) at 98°C for 30 minutes. Subsequently,
the sections were brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% H2O2 and Fc blocking by a specific protein block (Novocastra,
UK) the samples were incubated with the primary antibodies at room temperature. Staining was revealed by polymer detection kit (Novocastra) and DAB (3,3′-diaminobenzidine, Novocastra, Leica Biosystems Inc.) substrate-chromogen. The slides were counterstained with Harris hematoxilin (Novocastra).
Samples were stained with an anti-human IL-23 (Polyclonal, 1:100, pH 6, AbD Serotec, Bio-Rad Laboratories S.r.l.), or anti-IL-23R (Polyclonal, 1:100, pH 6 Millipore Cat.#06-1331) followed by a second layer antibody followed by polymer detection kit. The sections were analyzed under a Leica DMD108 optical digital microscope (Leica Microsystems)
IF staining was performed as detailed above for IHC. The following primary and secondary antibodies were used: anti-IL-23 (Polyclonal, 1:100, pH 6, AbD Serotec), IL-23R (Polyclonal, 1:100, pH 6, Millipore Cat.#06-1331), IL-12Rβ1 (Polyclonal, 1:50, pH 9, Novus Biologicals NBP1-49875), CD20 (Clone L26, 1:100, pH 9, Novocastra, Leica Biosystems), CD40L (Polyclonal, 1:250, pH 9, Abcam ab65854), CD68 (Clone KP-1, 1:50, pH 9, DakoCytomation). After Fc blocking, primary antibody binding was revealed by secondary antibodies either conjugated with Alexa Fluor 488 or Alexa Fluor 568. The slides were counterstained with DAPI stain (all Invitrogen Molecular Probes, ThermoFischer Scientific). All the IF-stained sections were analyzed under a Zeiss Axioscope A1 optical microscope equipped with an IF module (Zeiss, Oberkochen, Germany) and microphotographs were collected using a Zeiss Axiocam 503 color digital camera (Zeiss).
Double-marker IF staining was performed by two sequential rounds of single-marker IF staining. In the case of primary antibodies from the same source, primary and fluorochrome-conjugated secondary antibodies were pre-incubated together in vitro, prior to incubation onto
sections, to avoid cross-binding of the secondary antibodies as detailed in ref. (52). This technique was applied only in double-marker IF staining with anti-IL-23 and anti-IL-23R antibodies.
Xenograft studies
Six-to-eight week old female NOD/Shi-scid,γcnull (NSG)mice (The Jackson Laboratory, Bar Harbor, Maine, USA), a xenograft model for CLL growth in vivo (34), were housed in sterile enclosures under specific pathogen-free conditions. All procedures were performed according to the current International regulations and reviewed by the Animal Welfare Body of the IRCCS-AOU San Martino-IST National Cancer Research Institute, Genoa, Italy.
The procedures for xenografts have been reported in detail previously (35). Briefly, 50×106 PBMCs were infused by intravenous injection (IV) in each NSG mouse and engraftment
was verified by determining human circulating CD19+CD5+ leukemic cells starting at three weeks after inoculation.
To test the therapeutic effect of anti-IL-23 antibody, mice were treated intraperitoneally with anti-Human IL-23p19 Functional Grade Purified neutralizing IgG1 kappa monoclonal antibody (clone HNU2319, eBioscience) (36). Mice received 4 injections of a 20 µg dose of the antibody, each dose being administered every second day beginning with the week following engraftment, while the control group was similarly treated with mouse IgG1 kappa isotype control (clone P3.6.2.8.1 Functional Grade Purified, eBioscience) antibody, Three days after the last dose of antibody, animals were sacrificed in a saturated CO2 chamber and autopsies were
performed. Blood, bone marrow flushing, and sections of spleens and livers were designated for flow cytometry, IHC and IF in situ analysis as described above.
Formalin-fixed and paraffin-embedded spleen and liver specimens were analyzed for the presence of human CLL infiltrates and for the presence of co-injected bystander T cells by single- and double-marker IHC and IF as detailed above. The primary antibodies used were: anti-CD20 mouse monoclonal antibody (clone L26) ; anti-CD4 rabbit monoclonal antibody (clone SP35), and anti-CD8 rabbit monoclonal antibody (clone SP57) (Ventana Medical Systems, Roche). The sections were deparaffinized and antigen-retrieval was performed with citrate buffer high pH for 8 min. Then mAbs were incubated for 30 min at 37°C followed by addition of the polymeric detection system Ultraview Universal Red Detection Kit (Ventana Medical Systems).
An appropriate positive tissue control was used for each staining run; the negative control consisted of performing the entire IHC procedure on adjacent sections in the absence of the primary antibody. The sections were counter-stained (automatically) with Gill’s modified hematoxylin and then cover-slipped. The sections were evaluated by two observers with an Olympus light microscope using 10X, 40X, and 63X objectives.
Double staining with CD20 and Ki67 by IHC was performed by incubation at 37°C with a specific anti-human Ki67 antibody (MIB-1, DakoCytomation, dilution 1:25) and followed by addition of the polymeric detection system Ultraview Universal DAB Detection Kit (Ventana Medical Systems, Roche). The automated program provides dispensing of the second antibody (anti-CD20, L26-Ventana Medical Systems, Roche), followed by addition of the polymeric detection system Ultraview Universal RED Detection Kit.
CD16 IHC was performed following antigen retrieval using a pH 9 pre-treatment solution (Leica Biosystem), through overnight incubation with a primary monoclonal antibody specifically recognizing human CD16 (clone 2H7, Leica Biosystems, dilution 1:80). The binding of the primary antibody was revealed by a polymer detection method (Novolink, Leica
Biosystems) using the AEC chromogenic substrate. The slides were counterstained with Harris hematoxylin (Novocastra).
Murine xenografts were analyzed for the in situ expression of IL-23R, IL-12Rβ1, IL-23, CD20, and Ki67 (Clone MIB-1, 1:75, pH 6, DakoCytomation) by IF. After Fc blocking, binding of primary antibodies was revealed by fluorochrome-conjugated secondary antibodies: Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L), Alexa Fluor 488-conjugated donkey anti-goat IgG (H+L), Alexa Fluor 568 goat anti-rabbit IgG (H+L), Alexa Fluor 568 goat anti-mouse IgG (H+L), and Alexa Fluor 568 donkey anti-goat IgG (H+L). The slides were counterstained with DAPI Nucleic Acid Stain (Invitrogen Molecular Probes, Thermo Fisher Scientific). All the sections were analyzed under a Leica DM3000 optical microscope and microphotographs were collected using a Leica DFC320 digital camera (Leica Microsystems).
Fresh spleen and liver tissue samples from xenografted NGS mice were mechanically resuspended with gentleMACS™ Dissociator (Miltenyi Biotec GmbH,). Previous to mechanical resuspension, the spleens were enzymatically digested using The Spleen Dissociation Kit (Miltenyi Biotech GmbH). The single-cell suspensions were evaluated by flow cytometry analysis with a FACSCanto (BD Biosciences) using anti-human CD45 FITC, CD19 PECy7, CD5 APC, (BD Biosciences); anti-Human 12Rβ1 FITC (R&D Systems), anti-Human IL-23R-PE (R&D Systems), anti-Human CD19 APC-H7 (BD Biosciences), CD5 PerCP-Cy5.5 (BD Biosciences), CD45 APC (Miltenyi Biotech GmbH); Human IL-23p19 eFluor® 660, anti-Human IL-12/IL-23 p40 PerCP-eFluor® 710 (eBioscience), CD45 FITC, CD19 APC-H7 (BD Biosciences). Cell proliferation was assessed by Ki67 FITC (DakoCytomation), CD19 PE-Cy7, CD5 PE (BD Biosciences), CD45 APC (Miltenyi Biotech GmbH) staining, while apoptosis was evaluated by Annexin V FITC, CD19 PE-Cy7, CD5 PE, and CD45 APC.
In preliminary tests, purified CLL cells were activated in vitro by autologous activated T cells, purified by negative selection, and exposed to IL-23 (200ng/ml) for 10 minutes at room temperature. Since the CLL cells so treated express IL-23R complex and hence can bind IL-23, it was conceivable that the anti-23 antibody used for treatment could reveal the presence of IL-23 on CLL cell surface by flow cytometry. No staining was however observed. Likewise, CLL cells activated in vitro and exposed to IL-23 in vitro were not killed by the anti-IL-23 antibody used for treatment in the presence of complement (NSG mouse serum used at different dilutions 1:10, 1:20, 1:40). This indicates that the “cure” of NSG mice could not be attributed to direct cytotoxic effect on engrafted CLL cells by the inoculated antibody.
In specific experiments, as specified in the text, each NSG mouse received 100×106
PBMCs IV; following demonstration of engraftment two weeks later, therapy was administrated as described above. After 21 days of the final antibody dose, animals were sacrificed and tissues analyzed for the presence of neoplastic cells.
Depletion of IL-23R positive CLL subclones by magnetic beads
IL-23R positive cells were removed from the CLL clones using the MiniMACS system (Miltenyi Biotec GmbH). Briefly, the cells were exposed to IL-23R-PE monoclonal antibody (R&D Systems Inc., USA) followed by anti PE-coated magnetic micro-beads. The cells were passed first through a LD column designed for stringent depletion of unwanted cells (even weakly labeled cells) by retention of IL-23R positive cells; 99% of cells not retained by the column were IL-23R-negative. Subsequently, both depleted and non-depleted CLL cells were cultured in the presence of autologous T cells activated by CD3/CD28+IL-2, and the IL-23R complex on CLL cells was evaluated by flow cytometry at different time points up to 360 h.
Cell transfection
Silencer™ Select siRNA Negative Controls (n°1 and n°2 #4390844 and #4390847, Thermo Fisher Scientific), Silencer™ Select pre-designed for IL-23R chain (#s45268, #s45269, and #s45270, Thermo Fisher Scientific) and Silencer™ Select for IL-23p19 chain (IL-23A #28329, #28330, and #28331, Thermo Fisher Scientific) were delivered to CLL cells using the Neon Transfection System (Invitrogen, Thermo Fisher Scientific) at the final concentration of 5 nM/2×106 CLL cells as already described (4). Optimal transfection and survival of CLL cells
was obtained by applying 1 pulse at 2150 pulse voltage and a 20 pulse width, as indicated by the manufacturer for the primary blood-derived suspension cells protocol. After transfection, cell suspensions were transferred to a 24-well plate containing 500 μL of culture medium without antibiotics [RPMI -1640 with L-glutamine and 10% FBS (Gibco, Thermo Fisher Scientific), Na-pyruvate 0.1% (Euroclone)] at 37°C and incubated at the final concentration of 2×106 CLL
cells/mL/well in a 5% CO2 atmosphere incubator.
PCR analyses
Fragment length IGHV-IGHD-IGHJ analyses were performed by PCR amplification with primers specific for FR1-IGHV or FR3-IGHV gene subgroup specific primers in conjunction with 6-FAM-5’-CTTACCTGAGGAGACGGTGACC IGHJ gene-specific primer (53).
IL-12Rβ1 expression was evaluated by quantitative real time PCR (q-RT PCR) using TaqMan Assays (Applied Biosystems, Thermo Fisher Scientific, Massachusetts, USA) specific for IL-12Rβ1 (cod. Hs01106578) normalized versus POLR2A (cod. Hs00172187). All samples were analyzed at different time points and run in triplicate (duplicate for the POL2RA gene). Data of single patients were exported from StepOne 3.2 software (Applied Biosystems) and collectively analyzed by using DataAssist software (Applied Biosystems).
In siRNA transfection experiments, mRNA knock-down of IL-23A and IL-23R were measured at 6 h after transfection using TaqMan Gene expression Assay (Hs00332759_m1 for IL-23R and Hs00372324_m1 for Il-23A gene). The siRNA control transfected sample was used as calibrator. Data were normalized versus two different housekeeping genes (POL2RA cod. Hs00172187_m1 and HPRT1 cod. Hs99999909_m1, Applied Biosystems).
All Real time PCR experiments were performed using the TaqMan Universal Master Mix II according to the manufacturer’s instructions (Applied Biosystems) on the StepOne Plus PCR instrument (Applied Biosystems). Data were expressed as fold change according to the ΔΔCT formula.
Correlation with IL-23R expression and engraftment capacity by CLL cells
CLL cells from 21 cases with different expression of IL-23R side chain were categorized as positive (n=17) or negative (n=4) according to the calculated clinically-relevant cut-off (23%) as described in the Results section and Fig. 1. These cases (summarized inTable S4) were injected in NSG mice (2–4 replicates for each CLL). The IHC index is a measure of the spread of leukemia based on the average diameters of the follicular lesions. We assigned a numerical value of 1 to the follicles with diameters (±SD) of 102 (±90) × 42 (±7) µm; a value of 3 to follicles with a diameter 195 (±80) × 138 (±85) µm; a value of 6 to follicles between 399 (±245) × 300 (±39) µm, and a value of 12 to follicles between 734 (±461) × 540 (±167) µm. The IHC index is given by the sum of the number of follicles multiplied by the value assigned according to size as described previously (35).
Fig. S1. Gating strategy to analyze IL-23R complex induction after activation by CD40L-expressing fibroblasts. Experiments on 2 representative CLL cases CD40L-expressing low IL-23R (A and C) or high IL-23R (B and D). (A-B) Upon culture with Mock or CD40L- expressing fibroblasts (CD40L-TC), 2 CLL cell subpopulations could be distinguished based upon their SSC-A/FSC-A features. Viable cells (live cells) were double negative for Annexin V/PI staining. (C-D) Expression of CD80 and CD86 activation markers by purified CLL cells co-cultured for 48 h with Mock or CD40L-TC. Viable cells, identified based upon their SSC-A/FSC-A features were gated. Red lines indicate staining with the relevant mAb while gray lines indicate the control staining with an unrelated mAb. Relative fluorescence intensity (RFI) values reported are the ratios between the mean fluorescence intensity (MFI) of cells stained with CD80 or CD86 mAbs and the MFI of the isotype control antibody measured by flow cytometry.
Fig. S2. IL-12Rβ1 mRNA production after CLL cell activation by CD40L-expressing fibroblasts. IL-12Rβ1 mRNA expression was measured at different time points in CLL cells co-cultured with the indicated cells. The mRNA expression was calculated for each time point as fold change by using CLL cells cultured with Mock (not expressing CD40L) cells as calibrator normalized versus POL2RA gene mRNA. Results represent data from three different CLL patients.
Fig. S3. Expression of a complete IL-23R by CLL cells after depletion of IL-23R–positive cells and coculture with autologous activated T cells. CLL cells were evaluated for the expression of the complete IL-23R (top left). The cell suspensions were subdivided into 2 fractions only 1 of which was depleted of IL-23R-positive cells as depicted. The 2 cell fractions were cultured with CD3+CD28+ beads and IL-2 causing autologous T cells activation. After 120 h in culture, CLL cells were gated as CD5+CD19+ double positive cells and analyzed for the presence of the IL-23R complex.
Fig. S4. Demonstration of intracytoplasmic IL-23 chains in CLL cells. Purified CLL cells were incubated with CD40L-TC for 72 h. The cells were harvested and stained for the IL-23R complex and for intracytoplasmic IL-23 chains. IL-23R complex positive and negative cells were gated and analyzed for IL-23 expression as indicated. IL-23p19/p40 chains were found primarily within the IL-23R-complex expressing cells. Shown is a representative experiment of the three carried out on different CLL samples with identical results.
Fig. S5. HS5 stromal cells do not express CD40L. Flow cytometric analysis of HS5 stromal cell line.
Fig. S6. Enhanced IL-23R complex expression by coculture of PBMC with NLC. Three different cases of CLL were cultured in the indicated conditions before (unpurified CLL cells, PBMCs) or after removal of T cells and NK cells (purified CLL cells by negative selection). IL-23 receptor chains were then measured by flow cytometry.
Fig. S7. IL-23R complex expression by CLL cells cocultured with stromal cells or NLCs. (A) IL-23R complex measured on CLL cells following co-culture with HS5 stromal cells. (B) CLL cell spontaneous apoptosis in cultures with or without stromal cells. (C) IL-23R complex expression by purified CLL cultured in the presence or absence of Nurse-like cells (NLC). (D) Spontaneous apoptosis of CLL cells cultured with or without NLC. P values (Wilcoxon test) in A and C indicate comparison of CLL IL-23R complex expression versus control conditions, in B and D indicate comparison of CLL cell apoptosis versus control conditions. The asterisks P values statistically significant (P < 0.05)
Fig. S8. Relationship between the BCR signaling pathway and IL-23R complex expression in CLL cells. (A–B) Purified CLL cell suspensions were cultured with Dynabeads coated with anti-human IgM (G-Ab), in the presence or absence of IL-4 for 72 h and investigated for the expression of the IL-23R complex (A) and cell viability (B). Each dot represents a different CLL clone. (C-E) purified CLL cells were incubated with ibrutinib (1 M) 1 h before co-culturing for an additional 72 h with CD40L-TC or Mock cells and subsequently tested for IL-23R complex expression (C), phosphorylated BTK (pBTK) (D), and cell viability (E). The data in C-E refer to a representative test using a CLL patient sample (GE1-CC45). Similar results were observed for 2 other CLL cases.
Fig. S9. Silencing of IL-23A or IL-23R genes by siRNA. CLL cells were first stimulated by co-culture with CD40L-transfected fibroblasts for 72 h. CLL cells were then transfected with three different siRNA targeting the IL-23A gene (siRNA IL-23A), or three different siRNA targeting IL-23R (siRNA IL-23R) or two different siRNAs not targeting any gene product (siRNA neg-CTR). (A) Expression of IL-23A and IL-23R genes was evaluated in RD0468 CLL cells after 6 h of transfection with siRNAs. Data were obtained by using the ΔΔCT formula and by normalizing versus two different control genes (POL2RA and HPRT). Representative of n=2 independent experiments. After transfection, CLL cells were co-cultured with CD40L-TC for a further 24 h and analyzed for (B) cell viability as ANNEXIN-V/PI negative cells; (C) the proliferative capacity as Ki67 positive cells, and (D) IL-23 complex-expressing cell clone. The data in B-D refer to a representative test using a CLL patient sample (RD0468). Similar results were observed using two other CLL cases.
Fig. S10. STAT3 phosphorylation induced by CLL cell exposure to IL-23. (A-D) PBMC from CLL patient TG602 (48.2% IL-23R at T0) were cultured with CD3/CD28 beads and IL-2 or with medium alone (120 h). Subsequently, CLL cells were harvested, purified and briefly stimulated at 37°C with or without IL-23 (200 ng/mL) for the indicated times. (A and C) STAT3 phosphorylation (pSTAT3) in CLL cells not co-cultured with activated T cells (and express low IL-12Rβ1 chain levels as shown in A) after a brief exposure to IL-23 (200 ng/mL) and (B and D) in CLL cells following co-culture with CD3/CD28 beads and IL-2 inducing the expression of the complete IL-12Rβ1/IL-23R heterodimer (as confirmed by flow cytometry in B). (E-F) pSTAT3 induction in two other CLL cases stimulated with CD3/CD28 beads and IL-2 as above. CLL GE1-TM145 (E) and PG604 (F) were both IL-23R negative at T0.
Fig. S11. NSG mice engrafted with CLL cells. Immunohistochemistry analysis with anti-CD20 (left, magnification 40x) and confocal microscopy images of spleen sections stained with IL-23R and IL-23 as indicated (magnification 200x). Spleen sections were from NSG mice engrafted with CLL cases with different baseline levels of IL-23R expression: ZC0010 (8.1%), MG0248 (19.5%), and CD0310 (23.7%).
Fig. S12. Minimal residual disease detection in mice tissues after treatment with αIL-23p19. Minimal residual disease was investigated by fragment length analysis of BCR rearrangement gene in 2 representative mice injected with CLL PA0146 and treated with anti-IL23p19 or isotype control (see Fig. 7). Blue peaks indicated by the red arrows, show the presence of the BCR gene rearrangement of the leukemic clone in the indicated tissues.
Fig. S13. Anti–IL-23p19 mAb does not induce antibody-dependent cytotoxicity. (A) Immunohistochemistry analysis of human CD16 positive (NK) cells in sections of paraffin embedded spleen (CLL PA0146) from a mouse treated with isotype control (CTR) (left) or
αIL-23p19 (right) mAb (magnification 200x and box 630x). Arrows indicate the presence of few human CD16-positive cells in proximity or within leukemic nodules in NSG mice both treated with isotype CTR and αIL-23p19. (B) CLL cells from case RD0468 were stimulated by activated T cells for 8 days. CLL cells (CD19+CD5+) expressing at this time elevated levels of IL-23R complex, were purified by negative selection and incubated for 10 min at room temperature with an excess of IL-23 (200 ng/mL). Subsequently cells were incubated with isotype CTR (left) or αIL-23p19 neutralizing mAb for 30 min on ice, washed with cold PBS and incubated with goat anti mouse IgG conjugated with FITC secondary antibody for a further 30 min on ice. (C) CLL from case GE1-BA101 were stimulated as in (B), purified and incubated with IL-23 100 ng/mL followed by isotype CTR or αIL-23p19 neutralizing mAb for 2 h at 37°C in the presence of NSG mouse serum at different dilutions 1:10, 1:20, 1:40 used as source of complement. Cell viability was measured by Annexiv V and PI.
Fig. S14. Schematic representation of the IL-23R complex/IL-23 axis in CLL.Model of CLL cell activation leading to the induction of an IL-23R complex/IL-23 loop capable of supporting further CLL cell proliferation. Quiescent CLL cells, present in the circulation, express either the
IL-23R chain (IGHVunmutated) or no IL-23R chains (IGHV mutated). When the cells from both CLL subgroups enter the peripheral lymphoid tissues they are likely to encounter activated T cells as well as other cells expressing CD40L. This interaction invariably leads to the expression of a complete IL-23R and induces IL-23 secretion. The secreted IL-23 not only supports CLL cell proliferation, but also induces the maturation of certain T cells which may have influence on CLL clonal survival/expansion.
Table S1. Relationship between IL-23R chain expression and prognostic parameters.
Binet stage A patients were stratified into two groups designated as IL-23R-positive or -negative, based on the best-cut off value of 23%. This value could distinguish progressive from stable CLL cases by Receiver Operating Character (ROC) curve analysis (as shown in Fig. 1E). Significance was assessed by Fisher’s Exact test for all variables.
Prognostic parameters IL-23R negative (% of total) IL-23R positive (% of total) P-value CD38 Negative Positive Total 96 (85.0) 17 (15.0) 113 (100) 109 (77.9) 31(22.1) 140 (100) 0.1 ZAP-70 Negative Positive Total 56 (51.9) 52 (48.1) 108 (100) 67 (52.8) 60 (47.2) 127 (100) 0.49 IGHV Mutated Unmutated Total 85 (76.6) 26 (23.4) 111 (100) 94 (65.3) 50 (34.7) 144 (100) 0.049* IGHV CDR3 stereotypy No Yes Total 76 (72.4) 29 (27.6) 105 (100) 89 (74.8) 30 (25.2) 119 (100) 0.39 FISH Negative del(13)(q14) Trisomy 12 del(11)(q22·3) del(17)(p13.1) Total 38 (38.8) 50 (51.0) 6 (6.1) 4 (4.1) 0 (0.0) 98 (100) 40 (35.7) 48 (32.9) 13 (11.6) 9 (8.0) 2 (1.8) 101 (100) 0.22
Table S2. Univariate and multivariate bootstrapping validated Cox regression analysis of TTFT.
Variables
Bootstrapping Cox regression Analysis
Univariate Multivariate
HR (95% CI) P-value HR (95% CI) P-value
CD38-positive 3.92 (2.53–423) 0.001 1.48 (0.5–4.82) 0.4
Not cMBL 5.61 (2.97–16.76) 0.001 7.24 (2.59–465,602) 0.005
IGHV-unmutated 4.91 (3.18–7.8) 0.001 3.14 (1.04–12.88) 0.042
ZAP-70 positive 2.25 (1.44–3.56) 0.001 1.76 (0.65–6.04) 0.2
Table S3. Features of the CLL cases used in coculture experiments with CD40L-expressing fibroblasts (CD40L-TC).
ID IL-23R (%) IL-12Rβ1 (%) IL-23R
complex (%) IGHV IL-23R <23% FA0347 6.0 0.1 0.1 M MG0358 8.7 1.7 1.5 M RS0458 11.2 0.6 0.5 M IL-23R ≥23% VF0384 29.8 0.6 0.5 UM DA0059 33.3 11.2 5.3 M CM0289 40.6 1.4 1.2 UM AM0034 45.5 12.8 7.6 UM PF0481 46.7 14.7 8.7 M PT610 51.4 2.4 1.8 UM AR0242 70.6 0.1 0.1 UM MC0105 77.4 4.2 3.8 UM AG0318 80.9 0.0 0.0 UM MG0482 82.8 2.7 2.1 UM
Table S4. Summary of the features of all CLL cases whose cells were used for xenograft tests. The CLL clones were subdivided into IL-23Rhigh and IL-23Rlow according to the criteria described
above. The engraftment of each CLL clone was measured based upon the immunohistochemical index (IHC). Standard deviation refers to the mean of 2 to 4 engrafted NSG replicates for each CLL sample. CD38 and IGHV gene mutational status prognostic factors, TP53 mutational status, as well as cytogenetic abnormalities are provided.
ID IL-23R% IHC (mean±SD) CD38 IGHV TP53 del(13) (q14) +12 del(17) (p13.1) del(11) (q22.3) IL-23R <23% PM608 6.3 22±15 - M WT - - - - ZC0010 8.1 58±6 - M WT - - - - GM0041 13.91 137±41 - M WT + - - - GE1-BA101 15.6 ND - M WT - - - - MG0248 19.5 42±10 - M M + - - - IL-23R ≥23% CD0310 23.7 14±6 + UM WT + - - - GE2-RL201 25 82±30 - M WT + - - - RM0626 25.3 65±13 + UM M - - + - GD0051 25.78 23±3 - M WT + - - - VS0624 26.9 248±45 + UM WT - - - - PA0254 27.2 67±9 - M M + - - - SD36 28.6 40±8 + UM WT + - - + FP0499 28.79 137±37 + M WT + - - - PA0146 31.3 61±42 + M WT + - - - GC0626 33.0 23±8 + UM WT - - - + DA0346 33.48 17±1 - M WT + - - - DT0300 34.51 61±12 - M WT + - - - SG0232 36.78 39±3 - M WT + - - - PA0145 43.75 11±4 - UM WT - - - - MP0456 51.93 59±4 - M WT + - - - RD0468 52 56±6 + UM WT - + - - RD0296 58.91 30±0 - UM WT + - - -
