Advanced DMEM, IMDM, RPMI-1640, L-glutamine and penicillin-streptomycin (P/S), insulin-transferrin-selenium (ITS) were purchased from Gibco/Invitrogen, Waltham, Massachusetts, USA. StemSpan SFEM was purchased from STEMCELL Technologies StemCell Technologies, Vancouver, Canada. Phosphate buffered saline was purchased from Merck Life Science SL, Madrid, Spain. MSC osteogenic and adipogenic differentiation medium were purchased from Promocell, Heidelberg, Alemania. Human (h) Stem Cell Factor (SCF), hFMS-like tyrosine kinase 3 ligand (FLT3-L), human interleukin-3 (hIL-3), hIL-7 and hIL-15 were purchased from Miltenyi Biotec, Bergisch Gladbach, Germany. Nitro blue tetrazolium chloride (NBT), alizarin red, oil red-O, dexamethasone, L-phytohemagglutinin (PHA-L) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich, St. Louis, Missouri, USA. L-asparaginase was purchased from Jazz Pharmaceuticals Iberia SL, Barcelona, Spain. CellTrace Violet Cell Proliferation Kit and CellTrace CFSE Cell Proliferation Kit were purchased from Fisher Scientific SL, Madrid, Spain. Cell fixation and permeabilization Kit was purchased from Nordic-Mubio, Derio, Spain. Anti-CD3 (OKT3) and anti-CD28 (CD28.2) monoclonal antibodies (mAbs), 7-amino-actinomycin D (7-AAD), phycoerythrin (PE) annexin V apoptosis detection kit and fluorescein isothiocyanate, PE, peridin chlorophyll, allophycocyanin (APC)-, PE/ cyanine7 (PE/Cy7)-, brilliant violet 421 (BV421), BV510)-conjugated mAbs specific for human CD3, CD19, CD45, CD13, CD73, CD105, CD90, CD31, CD34 and isotype-matched negative control mAbs were purchased from BD Bioscience, Franklin Lakes, New Jersey, USA. APC-conjugated anti-Nestin mAb was purchased from R&D System, Abingdon, UK. The B-ALL cell lines SEM and NALM6 were obtained from DSMZ cell line bank, Braunschweig, Germany. Luciferase (Luc)/GFP-expressing-NALM6 cells were kindly provided by RJ Brentjens (MSKCC, New York, USA).

Peripheral blood (PB) mononuclear cells (PBMC) were isolated from buffy coats of healthy volunteers by Ficoll-Hypaque gradient centrifugation (GE Healthcare, Chicago, Illinois, USA).21 Buffy coats were obtained from the Catalan Blood and Tissue Bank (BST) on Institutional Review Board approval (HCB/2018/0030). BM B-ALL aspirates were obtained from pediatric patients with ETV6/RUNX1 or MLL-r B-ALL (table 1). BM aspirates from age-matched HD were obtained from the BST. Human samples were obtained after written informed consent in accordance with the Declaration of Helsinki.

Mononuclear cells (MC) from BM samples were isolated by centrifugation on Ficoll-Paque Plus density gradient. MCs were seeded at 2×10⁵ cells/cm² in advanced DMEM with 10% heat-inactivated FBS, L-glutamine and P/S (complete advanced DMEM medium) and maintained in a humidified atmosphere with 5% CO₂ at 37°C 2 days, after which non-adherent cells were collected and frozen and fresh medium was added. Adherent cells at >85% confluence were trypsinized and replated at 5×10³ cells/cm².22 The resulting BM-MSC cultures were maintained in complete Advanced DMEM medium. BM-MSC cultures were assessed daily for changes in growth rate and morphology. All experiments were performed with cells harvested between passage (P)2 and P5.

Growth kinetics of BM-MSC were measured as cumulative Population Doublings at each passage, as described.23 An equal number of BM-MSC (5×10³ cells/cm²) at P1 were initially plated for each patient/donor; cells were then harvested every 5 days, stained with trypan blue, counted and re-plated at 5×10³ cells/cm². The procedure was repeated until P6.

BM-MSC characterization was performed following the guidelines proposed by the International Society for Cellular Therapy (ISCT).24 The immunophenotype of cultured BM-MSC was assessed by FACS. A total of 1×10⁶ cells were incubated for 30 min at 4°C in the dark. Data acquisition and analysis were performed using a FACSCanto-II flow cytometer equipped with FACSDiva software (BD Biosciences). Differentiation was evaluated by plating BM-MSC in specific differentiation induction medium for 3–4 weeks. For osteogenic differentiation, BM-MSCs were stained to determine alkaline phosphatase (AP) activity using NBT and for calcium deposits with alizarin red.25 For adipogenic differentiation, BM-MSCs were stained with oil red-O to detect the lipid droplets.22 Differentiation capacity was further quantified by quantitative RT-PCR (qRT-PCR) and RNA was isolated from differentiated BM-MSC cultures using the Maxwell RSC simplyRNA Cells Kit (Promega, Madison, Wisconsin, USA). RT-PCR analysis was conducted using the SYBR Green PCR Master Mix (Fisher Scientific SL) with primers for specific adipogenic differentiation and osteogenic differentiation transcription factors. Analysis and mRNA quantification analysis were performed on the CFX384 Real-Time System (Bio-Rad Laboratorie, Hercules, California, USA). Reactions were performed in triplicate and gene expression values were normalized to those of HPRT. Gene-specific primer sequences for qRT-PCR are listed in online supplementary Table S1.

HD and B-ALL BM-MSC were seeded at 1×10⁴ cells/well in flat-bottom 96-well plates containing complete Advanced DMEM medium at 37°C for 24 hours. The next day, we evaluated T-cell proliferation using the CellTrace CFSE Cell Proliferation Kit. To do this, PBMCs were labeled with 5 µM CellTrace CFSE for 20 min at 37°C and then washed three times with RPMI 1640/1% heat-inactivated FBS.26 Subsequently, the BM-MSC culture medium was removed and CellTrace CFSE-labeled cells were added to the adherent BM-MSC culture at a BM-MSC:PBMC ratio of 1:5 and 1:10 in RPMI-1640 with10% heat-inactivated FBS and P/S (complete RPMI medium) (or plated alone), in the absence or presence of PHA-L (1 µg/mL). After 3 and 6 days of culture, non-adherent cells were collected, washed and stained with an anti-CD3 mAb and 7-AAD. Viable cells were gated as 7-AAD^-. Cell division was measured using dye dilution on the CD3⁺ population by FACS and the number of proliferating cells was determined by gating on the CD3⁺CFSE^low subset.26

Quantification of the proinflammatory cytokines IL-2, interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) was measured by ELISA using the OptEIA Human ELISA Kit (BD Biosciences) on supernatants from the aforementioned BM-MSC:PBMC cocultures. Unstimulated PBMC:BM-MSC cocultures were processed in parallel as a control. ELISA determinations were performed in triplicate.

HD and B-ALL BM-MSC were seeded at 1×10⁴ cells/well in flat-bottom 96-well plates containing complete Advanced DMEM medium at 37°C for 24 hours. The next day, the BM-MSC culture medium was removed and SEM cells (1×10⁵ cells/well) were added to the adherent BM-MSC culture at a BM-MSC:SEM ratio of 1:10 in IMDM/10% heat-inactivated FBS and P/S, in the absence or presence of dexamethasone (10 UI) or L-asparaginase (1 µM). Two days after, non-adherent cells were collected, washed and stained with an anti-CD19 mAb, 7-AAD and AnnexinV to determine cell apoptosis by FACS.27

We used our clinically validated pCCL second-generation lentiviral CD19-CAR backbone, which contains a human CD8 transmembrane domain and human 4-1BB and CD3ζ endodomains.28 We incorporated a GFP reporter gene after a 2A ribosomal skip sequence at the C-terminal CAR sequence to evaluate the transduction efficiency and the tracking of CAR expression. CAR-expressing viral particles pseudotyped with VSV-G were generated by transfection of HEK 293 T cells with pCCL, VSV-G and psPAX2 vectors using polyethylenimine (Polysciences, Warrington, Pennsylvania, USA). Supernatants were collected at 48 and 72 hours after transfection and concentrated by ultracentrifugation.

T-cells were activated by plate-coating with anti-CD3 and anti-CD28 mAbs in complete RPMI medium for 2 days and were then transduced with a CAR-expressing lentivirus at a multiplicity of infection of 10 in the presence of hIL-7 and hIL-15 (10 ng/mL).29 T-cells were expanded in complete RPMI medium plus hIL-7 and hIL-15 for up to 6 days. CAR transduction efficiency in T-cells was analyzed by FACS.

For cytotoxicity assays, BM-MSCs were seeded at 1×10⁴ cells/well in flat-bottom 96-well plates containing complete Advanced DMEM medium and incubated at 37°C for 24 hours. The next day, target cells (B-ALL cell lines or primary B-ALL cells, 1×10⁵ target cells/well) were incubated with CD19-CAR T-cells at an Effector:Target (E:T) ratio of 1:1, or alone, for the indicated periods in the absence/presence of BM-MSC. Cell lines were cultured in RPMI complete medium and primary cells were cultured in StemSpan supplemented with 20% heat-inactivated FBS, P/S, ITS, hSCF (100 ng/mL), hFLT3-L (100 ng/mL), hIL-3 (10 ng/mL) and hIL-7 (10 ng/mL). After 2 and 6 days of culture, non-adherent cells were collected, washed and stained with anti-CD3, anti-CD19, anti-CD10, anti-CD22 mAbs and 7-AAD. CAR T-cell-mediated cytotoxicity was determined by analyzing the residual living (CD10⁺CD22⁺7-AAD^-) target cells at each time point. BD TruCount absolute count tubes (BD Biosciences) were used for absolute cell counting. Quantification of the pro-inflammatory cytokines IL-2, IFN-γ and TNF-α was measured by ELISA using the OptEIA Human ELISA Kit (BD Biosciences) on supernatants harvested after 2 days of coculture at a 1:1 E:T ratio. ELISA determinations were performed in triplicate.

A well-established experimental mouse model for bowel inflammatory disease/acute colitis was employed.30 31 The in vivo procedure was approved by the Ethics Committee of the Spanish Council of Scientific Research. To induce acute colitis, 2,4,6-trinitrobenzene sulfonic acid (TNBS, 2 mg; Sigma) in 50% ethanol (100 µL) was administered intrarectally in male BALB/c mice aged 6–8 weeks (n=10/group). Control mice received 50% ethanol alone. At 4 hours after TNBS infusion, animals were treated intraperitoneally with medium alone, with 1×10⁶ HD BM-MSCs (n=3) or with 1×10⁶ B-ALL BM-MSC (MLL-r B-ALL n=3), resuspended in 200 µl of medium. Animals were monitored daily for the appearance of diarrhea, body weight loss, and survival. Colitis signs were scored based on stool consistency and rectal bleeding by a blinded observer using the following scale: 0=normal stool appearance, 1=slight decrease in stool consistency, 2=moderate decrease in stool consistency, 3=moderate decrease in stool consistency and presence of blood in stools, 4=severe watery diarrhea and moderate/severe blooding in stools. At day 9 or immediately after the death of the animal, the colons were collected and evaluated for macroscopic damage (scale 0–8) based on the grade of tissue adhesion, presence of ulceration and wall thickness in a blinded fashion by a researcher: ulceration (0=normal appearance, 1=focal hyperemia, no ulcers, 2=ulceration without hyperemia or bowel wall thickening, 3=ulceration with inflammation at 1 site, 4=two or more sites of ulceration and inflammation, 5=major sites of damage extending >1 cm along length of colon), adhesions (0=no adhesions, 1=minor adhesions, colon can be easily separated from the other tissues, 2=major adhesions); and thickness (maximal bowel wall thickness, in mm, measured with a caliper). PB was collected from the tail vein (day 4) and by cardiac puncture (day 9) and the levels of proinflammatory cytokines were determined in sera by specific sandwich ELISA using capture/biotinylated detection antibodies from PreproTech.

Ten-week-old non-obese diabetic Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were bred and housed under pathogen-free conditions. NSG mice were intratibial (IT)-injected with 1×10⁶ NALM6-Luc⁺ cells alone or together with 3×10⁵ BM-MSC,27 followed 4 days later by an intravenous infusion of 4×10⁶ CD19-CAR T-cells. Mice were followed up weekly by bioluminescence (BLI) using an in vivo imaging system (Lumina III; Perkin-Elmer, Wlatham, Massachusetts, USA). Mice were sacrificed at day 15 (controls) and day 35 (CAR-treated), and PB and BM samples were collected and analyzed by FACS to assess leukemic burden and CAR T-cell persistence.

Data were plotted as mean±SEM. One-way analysis of variance with Tukey’s post hoc test was used when three or more experimental groups were compared and Student’s paired t-test was used when two experimental groups were compared. All analyzes were performed with Prism software, V.8.0 (GraphPad Prism Software, San Diego, California, USA).

We isolated MSC from BM aspirates of 5 pediatric B-ALL, (2 ETV6/RUNX1 and 3 MLL-r), and we compared their morphology, immunophenotype, growth kinetics and differentiation potential with that of BM-MSC from three pediatric HD counterparts using the standardized criteria outlined by the ISCT.24 Table 1 shows the main biological and molecular/cytogenetic characteristics of B-ALL patients and HD. Adherent cell cultures were successfully established from all samples using standard MSC culture conditions.24 BM-MSC from both groups adhered to plastic and showed homogeneous spindle-shaped fibroblast-like morphology (figure 1A, online supplementary figure S1A). FACS analysis at P3 revealed that both BM-MSC groups display a bona fide MSC immunophenotype positive for CD105, CD90, CD73 and CD13 and Nestin, while negative for hematopoietic (CD34 and CD45) and endothelial (CD31) cell markers (figure 1B, online supplementary figure S1B).22 When we compared their expansion potential in vitro under identical culture conditions, HD BM-MSC showed a significantly superior proliferative potential than that of B-ALL BM-MSC (figure 1C). We next evaluated the differentiation potential of P3 BM-MSC cultures by exposing them to adipogenic and osteoblastic induction conditions. Both groups were similar with regard to adipogenic differentiation, measured qualitatively by the formation of lipid droplets detected by oil red-O staining; and to osteoblast differentiation, revealed by AP activity staining and by the presence of calcium deposits detected by Alizarin Red staining (figure 1D). Adipogenic and osteoblastic differentiation capacity was also confirmed quantitatively by qPCR, which showed a comparable and significant increase in the expression of early and late master regulator genes of adipogenic (CEBPα, PPAR-γ) and osteogenic (BMP2, RUNX2) differentiation (figure 1E).

MSCs are widely recognized for their immunomodulatory potential, including the inhibition of allogenic T-cell proliferation and the production of proinflammatory cytokines.19 20 We, therefore, monitored PHA-L-stimulated T-cell division in the absence or presence of BM-MSC in vitro. In line with published findings,32 33 we found that HD BM-MSC strongly inhibited T-cell proliferation in a dose-dependent manner (figure 2A). Of note, comparable inhibition of T-cell proliferation was exerted by B-ALL BM-MSC (figure 2A). We next analyzed these supernatants to test whether BM-MSC also regulate pro-inflammatory cytokine secretion. The analysis of supernatants showed that the levels of IL-2, IFN-γ and TNF-α were comparably and significantly lower in HD and B-ALL BM-MSC cocultures than in PBMC-only controls (figure 2B). Overall, these results show that both HD and B-ALL BM-MSC are equally immunosuppressive, as previously described.32 33

Having confirmed the immunosuppressive properties of B-ALL BM-MSC in vitro, we wished to test their ability to influence T-cell functions in vivo. To do this, we used a well-established preclinical model of acute colitis (figure 3A) that shares clinical, histopathological and immunological features with Crohn’s disease.30 31 As expected, TNBS-treated mice developed severe, acute illness characterized by substantial (~20%) and sustained body weight loss (figure 3B), bloody diarrhea, rectal prolapse and pancolitis, accompanied by extensive wasting syndrome (figure 3C), which caused 25% mortality over a 9-day period (figure 3D). Macroscopic examination of colons revealed profound signs of inflammation, hyperemia, ulceration and shortening (figure 3E). By contrast, mice that were treated with either HD or B-ALL BM-MSC were largely protected from colitis, as evidenced by a significant recovery of their body weight loss within 9 days (figure 3B), and by the improvement in the wasting syndrome and the signs of colon inflammation, which was reflected in the significantly lower mortality and colitis score as compared with control animals (figure 3C–E). In accord with their anti-inflammatory effects, treatment with either HD or B-ALL BM-MSC decreased the levels of the proinflammatory cytokines TNF-α and IL-6 in the sera of colitic mice (figure 3F). Collectively, our findings show that B-ALL BM-MSC can suppress inflammation in vivo to significantly protect mice against severe acute colitis.

While it is well established the contribution of BM-MSC in promoting survival, proliferation and chemotherapy resistance in B-ALL8–14 (online supplementary figure S2), the potential impact of B-ALL BM-MSC on CD19-CAR T-cell therapy has not been explored. Moreover, CAR T-cells are likely to experience the same loss of function/persistence as T-cells in an immunosuppressive B-ALL BMM. Consequently, we next explored the influence of BM-MSC on CD19-CAR T-cell activity. We first evaluated in vitro the impact of HD and B-ALL BM-MSC on CD19-CAR T-cell performance. We generated CD19-CAR T-cells following a standard protocol with anti-CD3/anti-CD28 plus hIL-7 and hIL-15,29 and studied the effects of BM-MSC on CD19-CAR T-cell cytotoxicity against NALM6 and SEM cells cultured at a 1:1 E:T ratio for 2 and 6 days (figure 4A). We found that the number of CD19-CAR-resistant NALM6 or SEM cells cultured in the presence of either HD or B-ALL BM-MSC was very similar to that of target cells exposed to CD19-CAR T-cells alone, a non-protective role of BM-MSC on NALM6 (figure 4B) and SEM (figure 4C) cell survival in CD19-CAR T-cell therapy, at least in vitro. We additionally studied the ex vivo impact of B-ALL BM-MSC on CD19 CAR T-cell cytotoxicity against autologous primary B-ALL blasts, which partially replicates the pathophysiological conditions of patients. We performed cytotoxicity assays using B-ALL blasts and patient-matched derived BM-MSC from five pediatric B-ALL patients (figure 5A). Irrespective of the difference in survival of B-ALL primary cells, after 2 days of culture the number of alive/resistant primary blasts from the five patients after CD19-CAR T-cell treatment was very similar in the absence or presence of autologous BM-MSC (figure 5B), consistent with the results obtained using B-ALL cell lines (figure 4). In addition, we found no significant differences in the levels of IL-2, IFN-γ and TNF-α in culture supernatants (figure 5C), confirming that BM-MSC from pediatric B-ALL do not promote resistance to CD19-CAR T-cells in vitro.

Finally, we evaluated the impact of HD or B-ALL BM-MSC on CD19 CAR T-cell cytotoxicity in vivo, using a mouse xenograft model with NALM6 cells. To recapitulate the interactions between the BM-MSC and leukemic cells, 1×10⁶ NALM6-Luc⁺ cells were IT injected into NSG mice alone or together with 3×10⁵ HD or B-ALL BM-MSC, followed 4 days later by 5×10⁶ CAR-T cells (figure 6A). Mice were followed up weekly by BLI. CD19-CAR T-cell-untreated mice showed massive leukemic burden and succumbed quickly to the disease (figure 6B and C). In contrast, mice treated with CD19-CAR T-cells, irrespective of whether they were transplanted or not with BM-MSC, similarly controlled the disease (figure 6B and C). FACS analysis of leukemic burden in PB and BM at day 15 confirmed the BLI data (figure 6D and E). All those mice treated with CD19-CAR T-cells with or without HD/B-ALL BM-MSCs in which the disease was controlled at the time of sacrificing the untreated controls (day 15) were followed up to 35 days, and found that neither HD nor B-ALL BM-MSC impaired long-term CD19-CAR T-cell activity (figure 6F,G). Collectively, neither HD nor B-ALL BM-MSCs apparently contribute to CD19-CAR T-cell resistance.