Bone marrow PDGFRα+Sca-1+-enriched mesenchymal stem cells support survival of and antibody production by plasma cells in vitro through IL-6

Bone marrow PDGFRα+Sca-1+-enriched mesenchymal stem cells support survival of and antibody... Abstract Plasma cells (PCs) acquiring long lifespans in the bone marrow (BM) play a pivotal role in the humoral arm of immunological memory. The PCs reside in a special BM niche and produce antibodies against past-encountered pathogens or vaccine components for a long time. In BM, cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4)-expressing PCs and myeloid cells such as dendritic cells are attracted to and held by CXC chemokine ligand 12 (CXCR12)-secreting stromal cells, where survival of the PCs is supported by soluble factors such as IL-6 and APRIL (a proliferation-inducing ligand) produced by neighboring myeloid cells. Although these stromal cells are also supposed to be involved in the support of the survival and antibody production, the full molecular mechanism has not been clarified yet. Here, we show that BM PDGFRα+Sca-1+-enriched mesenchymal stem cells (MSCs), which can contribute as stromal cells for hematopoietic stem cells, also support in vitro survival of and antibody production by BM PCs. IL-6 produced by MSCs was found to be involved in the support. Immunohistochemistry of BM sections suggested a co-localization of a minor population of PCs with PDGFRα+Sca-1+ MSCs in the BM. We also found that the sort-purified MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing, to which multiple roles in the in vitro PC support could be attributed. bone marrow niche, immunological memory, long-term antibody secretion Introduction Immunological memory is a highly integrated system composed of the coordinated functions of memory T and B cells as well as long-term antibody-secreting plasma cells (PCs) and the survival niches for those cells. Long-lived PCs maintain the humoral arm of the memory of previously experienced pathogens as well as vaccine components, so they provide effective secondary immune responses by having long lives in a special niche (1). It is important to clarify the molecular mechanism underlying the long life of PCs in a niche, because we do not have effective tools for regulating the magnitude and duration of antigen-specific antibody memory, which will be particularly important for eliminating harmful memories such as those established in allergies and autoimmune diseases. B cells can differentiate into two kinds of memory cells, namely memory B cells and PCs when stimulated by antigens and cytokines with cognate T-cell assistance. PCs then move to appropriate niches such as bone marrow (BM), acquire long-life ability and survive for several decades with continuous production of antibodies (2). The PCs in BM express cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4) and are located near the stromal cells expressing the CXCR4 ligand, CXC chemokine ligand 12 (CXCL12) and vascular cell adhesion molecule-1 (VCAM-1) (3). Other CXCR4+ cells such as eosinophils, basophils, megakaryocytes, dendritic cells and regulatory T cells also cluster with the CXCL12+ stromal cells, thus forming a survival environment for PCs (4–9). These clustering cells secrete factors contributing to the survival of and antibody production by PCs, such as IL-6, a proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), tumor necrosis factor α (TNFα) and CXCL12 (10–12). Co-culturing experiments on BM stromal cells and PCs in vitro also indicated that these cells maintain the longevity of PCs (13). These preceding studies indicated that various kinds of cells and molecules affect the survival and functions of long-lived PCs. However, the mechanisms as well as precise nature of the stromal cells have not been fully characterized yet. In BM, mesenchymal stem cells (MSCs) comprise a very small population accounting for only 0.05–0.08% of the total mononuclear cells (14). The MSCs in BM are multipotent cells able to differentiate into adipocytes, osteocytes and myocytes (15). On the other hand, MSCs in BM can have immunosuppressive functions, such as repression of proliferation of and cytokine production by T cells (16, 17), inhibition of activation and proliferation of B cells and their differentiation into PCs (18–21). In addition, Nestin+ MSCs form a niche for maintaining hematopoietic stem cells (HSCs) in BM (22). Although MSCs occupy only a small compartment in BM, we hypothesized that these cells might also play a role in the maintenance of PCs in BM, on the basis of the knowledge of their immunosuppressive functions as well as a role for HSCs. The conventional method for isolation of MSCs from BM cells is based on their adhesive nature as to plastic dishes (23). Since this isolation protocol takes several weeks, MSCs tend to differentiate into other types of cells (24). In addition, MSCs prepared by this protocol are heterozygous and possibly include HSCs and other adherent cells (25, 26). In order to test our hypothesis as to MSCs’ potential role in maintenance of BM PCs, we considered that choosing a suitable method for purifying MSCs was important. Houlihan et al. (14) established a means of flow cytometric isolation of MSCs using cell-surface MSC-specific markers, platelet-derived growth factor receptor alpha (PDGFRα) and stem cell antigen-1 (Sca-1), which enables us to obtain highly purified MSCs. By using this protocol, we expected that our hypothesis could be reasonably verified under conditions minimizing any effect of contaminating cells in the MSC preparation. In this study, we sort-purified PDGFRα+Sca-1+ MSCs from BM cells employing the flow cytometric protocol, co-cultured them with CD138+B220− PCs also isolated from BM cells by flow cytometry and evaluated the PC survival and antibody production by enzyme-linked immunosorbent spot (ELISpot) formation and enzyme-linked immunosorbent assaying (ELISA). We found that soluble factors including IL-6 contribute to the survival of and antibody production by PCs. We also showed that the isolated MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing (scRNA-seq), suggesting multiple roles are played by different groups of the MSCs. Methods Mice C57BL/6 (B6) mice were purchased from CLEA Japan (Shizuoka, Japan), and IL-6−⁄− mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The Blimp1-GFP transgenic mice (27) were kept in a B6 genetic background. Mice were maintained and bred in the animal facility of the Institute of Development, Aging and Cancer, Tohoku University, an environmentally controlled and specific pathogen-free facility, according to the guidelines for experimental animals defined by the university, and all animal experiments were approved by the Tohoku University Animal Studies Committee. All experiments were performed on 6- to 32-week-old male and female mice. Antibodies and flow cytometry Allophycocyanin (APC)-labeled rat anti-mouse PDGFRα (Clone APA5), APC-labeled rat anti-mouse CD138 (281-2), FITC-labeled rat anti-mouse Sca-1 (D7), PE-labeled rat anti-mouse CD45 (30-F11), PE-labeled Armenian hamster anti-mouse/rat CD29 (HMβ1-1), PE-labeled Armenian hamster anti-mouse/rat CD49e (HMα5-1), PE-labeled rat anti-mouse CD90.2 (53-2.1), PE-labeled rat anti-mouse CD105 (MJ7/18) and PE-labeled rat anti-mouse TER-119 (TER-119) were purchased from BioLegend (San Diego, CA, USA). PE-labeled rat anti-mouse CD44 (IM7) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Cells were incubated with staining reagents in staining buffer [1% bovine serum albumin (BSA) in PBS] for 30 min at 4°C. Cells were acquired with a FACS Aria III using FACS Diva software (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Cell isolation by flow cytometry PDGFRα+Sca-1+CD45−TER-119− MSCs were isolated from femoral and tibial BM of 6–10-week-old mice with a FACS Aria III (BD Biosciences) according to the established method (14). CD138+B220− PCs were isolated from femoral and tibial BM of 26–32-week-old mice by flow cytometry. Cell culture Isolated MSCs were suspended at a density of 3 × 104 cells ml−1, and a 100 µl aliquot was applied to each well of 96-well plates. Three days later, sort-purified PCs suspended at 8 × 104 cells ml−1 were seeded at 100 µl per well for the MSC culture. The culture was conducted in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) containing penicillin/streptomycin (Sigma-Aldrich), 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 10 mM HEPES (Dojindo Laboratories, Kumamoto, Japan), 1 mM sodium pyruvate (Sigma-Aldrich), 0.1 mM MEM-NEAA (Thermo Fisher Scientific) and 50 µM 2-mercaptoethanol (Sigma-Aldrich) in a humidified atmosphere of 95% air and 5% CO2 at 37°C for the indicated period of time. For assessing a role of IL-6, recombinant mouse IL-6 (Wako Pure Chemical Industries, Osaka, Japan) and/or LEAF purified anti-mouse IL-6 antibody (BioLegend) were added to culture as indicated. Generation of BM-derived cultured dendritic cells BM cells prepared from femoral and tibial BM of B6 mice were cultured with 20 ng ml−1 GM-CSF (Peprotech, Rocky Hill, NJ, USA) for 7 days (28) to obtain BM-derived cultured dendritic cells (BMDCs). At day 6, lipopolysaccharide (Sigma-Aldrich) was added at 1 µg ml−1 to promote maturation. ELISpot assaying For detection of IgG-secreting cells, Elispot MultiScreenHTS Filter Plates (Merck Millipore, Billerica, MA, USA) were coated with 10 µg ml−1 affinity-purified goat anti-mouse IgG-Fc antibodies (Bethyl Laboratories) and then incubated overnight at 4°C. The plates were blocked with RPMI culture medium for 90 min. Freshly sort-purified or cultured PCs were added at a volume of 200 µl per well, followed by incubation at 37°C. After 4-h incubation, the plates were treated with HRP-conjugated goat anti-mouse IgG-Fc detection antibodies (Bethyl Laboratories) and developed with 3-amino-9-ethylcarbazole. Spots were examined under an upright microscope (BX53, Olympus, Tokyo, Japan) and analyzed with ImageJ 1.42q software. The cell survival rate was calculated from the spot number after day 7 or day 14 culture compared with that on day 0. Microscopic analysis Images of immunofluorescently labeled cells were acquired on a fluorescence microscope BZ-X700 (Keyence, Osaka, Japan), and analyzed with BZ-X analyzer (Keyence) and ImageJ software. Numbers of GFP+ PCs and propidium iodide (PI)+ PCs were counted automatically by BZ-X analyzer (Keyence). After automated enumeration of PI+ cells with the BZ-X analyzer, we excluded the counts for the cells larger than 400 µm2, which was the maximum area for PCs observed in the culture without MSCs. Then, the cells having apparently abnormal shapes being different from that of typical PCs were visually excluded. Immunohistochemical analysis Mouse femurs were isolated and frozen BM sections were prepared according to the Kawamoto’s film method. Frozen blocks were cut into 5-µm sections, using a Microm HM505E cryostat, and fixed in 4% paraformaldehyde for 10 min. After blocking with 2% BSA–PBS with 5% rat serum (Wako) for 1 h, samples were incubated at 4°C overnight with biotin-labeled anti-PDGFRα antibody (1:40, BioLegend), Alexa Fluor 647 anti-Sca-1 antibody (1:50, BioLegend), GFP-tag polyclonal antibodies (1:1000, Thermo Fisher Scientific) in 1% BSA–PBS. The secondary antibody reaction for biotinylated PDGFRα was streptavidin Alexa Fluor 594 antibody (1:500, Invitrogen), and for GFP was Alexa Fluor 488 anti-rabbit IgG antibody (1:1000, Invitrogen) at room temperature for 1 h. ELISA Culture supernatants were frozen at −80°C until analyzed by ELISA. For detection of total IgG and IgM secreted by PCs, mouse IgG and IgM ELISA Quantitation Sets (Bethyl Laboratories) were used. For detection of IL-6, a Mouse IL-6 ELISA MAX™ Standard Set (BioLegend) was used. For detection of CXCL12, Quantikine ELISA Mouse CXCL12/SDF-1α (R&D Systems) was used. Each assay was performed according to the manufacturer’s instructions. Real-time PCR for quantitation of IL-6 mRNA RNA was isolated using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized using a ReverTra Ace PCR RT Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The cDNA was analyzed by quantitative PCR with SYBR Green Realtime PCR Master Mix Plus (Toyobo) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Primer sequences for Il6 were: forward, 5′-AGCCAGAGTCCTTCAGAGAGAT-3′; reverse, 5′-GAGAGCATTGGAAATTGGGGT-3′; for actb were: 5′-CGTTGACATCCGTAAAGACCTC-3′; reverse, 5′-AGCCACC GATCCACACAGA-3′. Transwell assaying Transwell cultures were set up in 24-well plates. ThinCert™ cell culture inserts with 0.4 µm-pore membranes of 1 × 108 cm−2 pore density (Greiner Bio-One, Kremsmunster, Austria) were used to physically separate PCs from MSCs. Plasma cells (8 × 103 cells per well) were cultured in the upper chamber, and MSCs (1 × 104 cells per well) in the lower chamber. Cytokine array A Mouse XL Cytokine Array Kit (R&D Systems) consisting of a total 111 different cytokine and chemokine antibodies spotted in duplicate onto the membrane was used according to the manufacturer’s directions. The membranes were developed with an ImageQuant LAS 4000 mini (GE Healthcare Japan, Tokyo, Japan) and analyzed with an ImageQuant TL (GE Healthcare Japan). Single-cell RNA sequencing Single-cell RNA sequencing (scRNA-seq) was performed with multiplex linear amplification2 (CEL-Seq2) as described previously (29). An MSC was sorted into a well containing 1.2 µl primer mix in a 96-well plate. After cell disruption at 65°C for 5 min, reverse transcription was performed at 42°C for 1 h with 0.4 µl first strand buffer, 0.2 µl 0.1 M DTT, 0.1 µl RNase Inhibitor and 0.1 µl Superscript II. The reverse transcript was inactivated by heating the mixture at 70°C for 10 min. The second strand reaction was performed at 16°C for 2 h with 7 µl H2O, 2.31 µl second strand buffer, 0.23 µl dNTP, ligase 0.08 µl, Escherichia coli DNA polymerase and 0.08 µl RNaseH. Double-strand DNA (dsDNA) samples in a 96-well plate were collected and cDNA cleanup was performed by the method described previously (29). In vitro transcription was performed at 37°C for 13 h with 3.2 µl ATP, 3.2 µl GTP, 3.2 µl CTP, 3.2 µl UTP, 3.2 µl 10 × T7 buffer, 3.2 µl T7 enzyme and 12 µl dsDNA. RNA samples were subjected to EXO-SAP treatment, RNA fragmentation, RNA cleanup and library preparation for Illumina RNA-sequencing as previously described (29). The libraries were sequenced on HiSeq2500, controlled by HiSeq Control Software v2.2.58, with 15 bases for read 1 and 36 bases for read 2. Gene expression analysis Expression analysis was performed using the CEL-Seq-2 pipeline (https://github.com/yanailab/CEL-Seq-pipeline) (29). Briefly, paired-end fastq files for each RNA-seq sample were demultiplexed to generate 96 single-end fastq files, each of which represents expression from a single cell. The single-end reads were of 35 bases and attached to 5-base unique molecular identifiers (UMI). The reads were mapped on the mouse GRCm38 reference genome using the Bowtie2 program. Gene expression was quantified with the SAM files as the number of reads mapped on the exonic region of each gene according to the RefSeq gene annotation on GRCm38. UMI was used to remove the PCR duplication biases. Statistical analysis Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA) on the basis of the results of at least three independent experiments. Significance was assessed by the unpaired two-tailed Student’s t-test, one-way analysis of variance (ANOVA) or two-way ANOVA. P <0.05 was considered statistically significant. Results MSCs support the survival of and antibody production by BM PCs To investigate the effect of MSCs on the survival of and antibody production by PCs, we first isolated CD45−TER-119−PDGFRα+Sca-1+ MSCs (14) from BM cells of wild-type C57BL/6 mice using a flow cytometer and then cultured them in vitro without PCs (Fig. 1A). Twenty-two days later, we characterized the surface markers on cultured MSCs, and found that the MSCs maintained the expression of PDGFRα and Sca-1 (Fig. 1B). The expression of other MSC markers, CD44, CD29, CD90 and CD49e, was also positive, while CD105 expression was bi-phasic, the CD105low population comprising the majority (Fig. 1C). On BM stromal cells, it was shown that the expression of CD105 decreased depending on the passage and cell density (30), suggesting that also on BM MSCs the CD105 expression decreased during the culture. To examine this possibility, we measured the CD105 expression on MSCs on the first day of isolation, and found that it was substantially positive on the freshly isolated MSCs (Supplementary Figure S1). Overall, we concluded that the isolated MSCs had a similar expression profile of surface markers to that reported previously (14). Fig. 1. View largeDownload slide Isolation of MSCs and PCs among BM cells by flow cytometry. (A) Flow cytometric isolation of MSCs. After exclusion of dead cells by PI staining, CD45−TER-119−PDGFRα+Sca-1+ cells were gated and sort purified as MSCs. (B, C) Surface marker expression by MSCs at day 22 of culture (solid line). Dotted line indicates isotype control. (D) Flow cytometric isolation of PCs among BM cells. CD138+B220− cells were sort purified after exclusion of dead cells by PI staining. Fig. 1. View largeDownload slide Isolation of MSCs and PCs among BM cells by flow cytometry. (A) Flow cytometric isolation of MSCs. After exclusion of dead cells by PI staining, CD45−TER-119−PDGFRα+Sca-1+ cells were gated and sort purified as MSCs. (B, C) Surface marker expression by MSCs at day 22 of culture (solid line). Dotted line indicates isotype control. (D) Flow cytometric isolation of PCs among BM cells. CD138+B220− cells were sort purified after exclusion of dead cells by PI staining. Next, we sort-purified CD138+B220− PCs from BM cells (Fig. 1D) and plated them onto a day 3 MSC culture. We employed BMDCs and BM stromal cell line OP9 as positive controls, because BMDCs and BM stromal cells were shown to support the survival of and antibody secretion by BM PCs (8, 13, 31). BM PCs were also cultured alone as a negative control. After 7 days of co-culture, the culture supernatant was collected and subjected to measurement of IgG and IgM by ELISA. Also, PCs were recovered from the co-culture, the IgG-secreting cells among them being enumerated by ELISpot assaying, and then positive cell numbers were compared with those beforehand on day 0 as a measure of the survival rate of antibody-secreting cells. We found that the number of functionally surviving, antibody-secreting PCs was two to three times higher in the MSC co-culture than among PCs cultured alone as assessed by ELISpot (Fig. 2A, left graph). We also measured the individual spot areas as a measure for the antibody secretion activity of PCs and found that it was significantly increased in the MSC co-culture (Fig. 2A, right graph), suggesting that the activity was higher in the PCs under co-culture than those cultured alone. Fig. 2. View largeDownload slide Effect of co-culture of BM PCs with MSCs on their survival and antibody production. (A) Quantification of functionally surviving cells by counting IgG-secreting PCs by means of ELISpot after culturing with (right picture) or without (left picture) MSCs for 7 days. Survival rate % (left graph) is calculated by comparison to the ELISpot count at day 0 culture, and spot area (right graph) is measured by ImageJ. Values are expressed as the mean + SD for four independent experiments performed in duplicate. (B) Estimation of survival of PCs in culture under a fluorescence microscope. Representative fields are shown. Live PCs cultured alone or with MSCs for 7 days were visualized as to GFP expression. Original magnification, ×20. Each signal of GFP+ PCs and PI-stained cells was counted, and live cell % was calculated as follows: GFP+ cell number ⁄ sum of GFP+ and PI+ cell number × 100. Values are expressed as the means + SD of three independent experiments. (C) Determination of IgG (left) and IgM (right) concentrations by ELISA in day 7 culture supernatants of PCs co-cultured with or without MSCs, OP9 or BMDCs. The data are shown as fold changes as to PCs without co-culture (designated as 1). Values are expressed as means + SD for three independent experiments performed in duplicate. (D, E) Time course of the survival rate (D), and IgG and IgM levels (E) in culture supernatants of PCs co-cultured with or without MSCs for up to day 14. Values are expressed as means ± SD for three or four independent experiments performed in duplicate. Statistical differences were analyzed by means of the one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. 2. View largeDownload slide Effect of co-culture of BM PCs with MSCs on their survival and antibody production. (A) Quantification of functionally surviving cells by counting IgG-secreting PCs by means of ELISpot after culturing with (right picture) or without (left picture) MSCs for 7 days. Survival rate % (left graph) is calculated by comparison to the ELISpot count at day 0 culture, and spot area (right graph) is measured by ImageJ. Values are expressed as the mean + SD for four independent experiments performed in duplicate. (B) Estimation of survival of PCs in culture under a fluorescence microscope. Representative fields are shown. Live PCs cultured alone or with MSCs for 7 days were visualized as to GFP expression. Original magnification, ×20. Each signal of GFP+ PCs and PI-stained cells was counted, and live cell % was calculated as follows: GFP+ cell number ⁄ sum of GFP+ and PI+ cell number × 100. Values are expressed as the means + SD of three independent experiments. (C) Determination of IgG (left) and IgM (right) concentrations by ELISA in day 7 culture supernatants of PCs co-cultured with or without MSCs, OP9 or BMDCs. The data are shown as fold changes as to PCs without co-culture (designated as 1). Values are expressed as means + SD for three independent experiments performed in duplicate. (D, E) Time course of the survival rate (D), and IgG and IgM levels (E) in culture supernatants of PCs co-cultured with or without MSCs for up to day 14. Values are expressed as means ± SD for three or four independent experiments performed in duplicate. Statistical differences were analyzed by means of the one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Because recovery of the cultured PCs might not be quantitative, particularly in a co-culture with MSCs because of potential adherence to MSCs as well as to the culture plates, we also checked the survival of PCs in culture using a fluorescent microscopic technique. To this end, live PCs were visualized as their GFP expression driven by a Blimp1 promoter prepared from BM cells of BlimpGFP mice (27), while dead PCs were stained with PI, albeit PI+ MSCs were scarcely detected in our analysis. We enumerated live GFP+ PCs and dead PI+ PCs, calculated the PC survival % as the rate of GFP+ cells among total GFP+ plus PI+ cells, and found that the PC survival % was higher at day 7 co-culture with MSCs than that without MSCs (Fig. 2B), indicating that MSCs support the survival of PCs. On ELISA measurement, both the IgG and IgM levels in the culture supernatant were significantly higher in the PC–MSC co-culture than with PCs alone (Fig. 2C). PC co-culture with OP9 or BMDCs had a similar but not superior effect on the immunoglobulin production to that obtained for the co-culture with MSCs (Fig. 2C). To test whether this supportive effect of MSCs on maintenance of functional PCs observed after day 7 culture could be sustained for a longer period of time, the PC–MSC co-culture was extended up to day 14. We found that the functionally surviving cell rate observed at day 7 was still maintained at day 14. Likewise, the IgG and IgM levels in the culture supernatant were even higher at day 14 than those at day 7, indicating that functionally surviving PCs were mostly maintained from day 7 to day 14 (Fig. 2D and E). Collectively, these data indicated that MSCs supported the survival of and antibody production by BM PCs as efficiently as OP9 cells and BMDCs do. IL-6 produced by MSCs is a supporting factor for functional PC survival To elucidate the mechanism by which MSCs support the survival of and antibody production by PCs, we examined potential IL-6 secretion by MSCs, because IL-6 is known to support PC survival (10). Firstly, we measured expression of IL-6 mRNA by RT–PCR. We isolated MSCs, PDGFRα+Sca-1– (PDGFRα+) and PDGFRα–Sca-1+ (Sca-1+) cells and cultured for 7 days with or without PCs. IL-6 mRNA levels in MSCs were significantly higher than PDGFRα+ cells and Sca-1+ cells (Fig. 3A). In BM stromal cells, expression of IL-6 mRNA was shown to be up-regulated upon culture with PCs (13), suggesting a potential signal from PCs to MSCs that leads to IL-6 mRNA transcription. In our analysis, however, IL-6 mRNA was not induced by co-culture with PCs. Therefore, MSCs constitutively express IL-6 mRNA, which could be a unique characteristics of MSCs. Next, we measured the IL-6 concentration in the supernatant of BM PCs co-cultured with MSCs, OP9 or BMDCs for 7 days and found that MSCs and BMDCs but not PCs or OP9 secreted IL-6 (Fig. 3B, left). The IL-6 level in the supernatant was comparable to those between day 7 and day 14 PC–MSC co-culture (Fig. 3B, right). These results suggested that IL-6 could be a supporting factor for PC survival provided by MSCs and BMDCs in our culture system, albeit that the mechanism for PC support by OP9 would not be dependent on IL-6. To verify the roles of IL-6 and other unidentified factors in functional PC survival, we examined the effect of addition of mouse recombinant IL-6 (rIL-6) and neutralization of MSC-derived IL-6 by anti-IL-6 antibody on the functional survival of PCs. IgG levels were increased but they did not exceed the levels of MSC co-culture (Fig. 3C), suggesting an involvement of other stimulating factor(s) in MSC co-culture. In the experiment of neutralization of IL-6 by anti-IL-6 antibody, IgG levels were decreased compared to the positive control MSC co-culture, but still higher than PC culture (Fig. 3C). These results suggest that IL-6 plays a role in supporting PCs, while other factors are also involved in the support. In addition, we co-cultured PCs with MSCs isolated from BM cells of Il6-deficient mice. ELISpot analysis revealed that the survival rate was reduced, and the IgG and IgM concentrations in the supernatant were markedly reduced but not completely abolished in the co-culture with IL-6-deficient MSCs (Fig. 3D). These results suggested that IL-6 is a substantial supporting cytokine for functional PC survival but not an exclusive factor. Fig. 3. View largeDownload slide Reduced functional survival of PCs co-cultured with IL-6-deficient MSCs. (A) Quantitative real-time PCR to determine the levels of IL-6 mRNA. MSCs, PDGFRα+Sca-1– (PDGFRα+) cells and PDGFRα–Sca-1+ (Sca-1+) cells were cultured for 7 days with or without PCs. (B) IL-6 concentrations in culture supernatants of PCs with or without co-culture at day 7 (left), and changes of the IL-6 levels in the supernatants of PCs up to day 14 (right). Values are expressed as means ± SD for three independent experiments. (C) IgG (left), IgM (middle) and IL-6 (right) levels in supernatants of PCs cultured alone and with MSCs treated with recombinant IL-6 (rIL-6, 10 ng ml−1) or anti-IL-6 antibody (αIL-6, 10 µg ml−1). Ctrl. means with or without isotype control. (D) Functional survival rate (upper left) of PCs and IL-6 (upper right), IgG (lower left) and IgM (lower right) levels in supernatants of PCs, cultured alone or co-cultured with MSCs prepared from wild-type or Il6-deficient mice. Values are expressed as means + SD of three independent experiments performed in duplicate or triplicate. Statistical difference was analyzed by one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. 3. View largeDownload slide Reduced functional survival of PCs co-cultured with IL-6-deficient MSCs. (A) Quantitative real-time PCR to determine the levels of IL-6 mRNA. MSCs, PDGFRα+Sca-1– (PDGFRα+) cells and PDGFRα–Sca-1+ (Sca-1+) cells were cultured for 7 days with or without PCs. (B) IL-6 concentrations in culture supernatants of PCs with or without co-culture at day 7 (left), and changes of the IL-6 levels in the supernatants of PCs up to day 14 (right). Values are expressed as means ± SD for three independent experiments. (C) IgG (left), IgM (middle) and IL-6 (right) levels in supernatants of PCs cultured alone and with MSCs treated with recombinant IL-6 (rIL-6, 10 ng ml−1) or anti-IL-6 antibody (αIL-6, 10 µg ml−1). Ctrl. means with or without isotype control. (D) Functional survival rate (upper left) of PCs and IL-6 (upper right), IgG (lower left) and IgM (lower right) levels in supernatants of PCs, cultured alone or co-cultured with MSCs prepared from wild-type or Il6-deficient mice. Values are expressed as means + SD of three independent experiments performed in duplicate or triplicate. Statistical difference was analyzed by one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MSCs secrete a number of soluble factors To explore the soluble factors other than IL-6 supporting the survival of and antibody production by PCs, we employed a cytokine array technique for identifying cytokines, chemokines, and growth factors in culture supernatants of MSCs and ones of OP9 for comparison (Fig. 4A and B). We identified CCL2, CHI3L1, IGFBP-2, -5 and -6, M-CSF, MMP3 and Pentraxin 3 as the soluble factors secreted specifically by MSCs but not OP9 (Fig. 4C). IL-6, detected on ELISA (Fig. 3B) and also as its mRNA expression (Fig. 3A), did not give a significant signal with the cytokine array technique (Fig. 4A), potentially because of an insufficient sensitivity of the antibody used in this array. To determine whether these MSC-specific factors have a supportive effect on PCs, we added some recombinant proteins to the PC cultures without MSCs. However, while IL-6, as a positive control, markedly sustained the IgG and IgM levels, the others did not (Supplementary Figure S2). Other soluble factors commonly detected in both MSCs and OP9, such as CXCL1, Cystain C, LIX, Osteopontin and Serpin E1, might also be candidate(s) for MSC-mediated PC support, which awaits further investigation. Fig. 4. View largeDownload slide Profiles of soluble factors in culture supernatants of MSCs and OP9 cells. (A, B) Detection of cytokines, chemokines and growth factors in culture supernatants of MSCs and OP9 by cytokine array analysis, in which the supernatants were blotted onto membranes in duplicate (A), followed by detection with each of the specific antibodies shown in B. (C) Quantification of signal intensities shown in A by measuring pixel density. The factors in boldface are those exhibiting notably higher expression in MSCs than OP9. The factors with asterisks are those tested for the supporting effect by the inclusion to PC culture shown in Supplementary Figure S2. Fig. 4. View largeDownload slide Profiles of soluble factors in culture supernatants of MSCs and OP9 cells. (A, B) Detection of cytokines, chemokines and growth factors in culture supernatants of MSCs and OP9 by cytokine array analysis, in which the supernatants were blotted onto membranes in duplicate (A), followed by detection with each of the specific antibodies shown in B. (C) Quantification of signal intensities shown in A by measuring pixel density. The factors in boldface are those exhibiting notably higher expression in MSCs than OP9. The factors with asterisks are those tested for the supporting effect by the inclusion to PC culture shown in Supplementary Figure S2. Examination of a role of MSC–PC direct contact in supporting PCs BM PCs express CXCR4 and are located near CXCL12+ stromal cells (3, 32). To determine whether MSCs express CXCL12, we performed ELISA assaying. CXCL12 was detected in the supernatant of the MSC–PC co-culture, but it was negligible in the PC culture without MSCs (Fig. 5A), suggesting a potential of MSCs for producing CXCL12, secreting it into the milieu, attracting PCs and interacting with those in close vicinity in BM. Therefore, next we were interested in examining a potential role of MSC–PC direct contact in supporting PCs. To this end, we employed a transwell culture where BM PCs were placed in the upper chamber separated from MSCs in the bottom chamber by a semi-permeable membrane, and measured the IgG and IgM levels in the culture supernatant after 7 days. We found that the IgG and IgM levels in the culture supernatant were decreased upon separation (Fig. 5B), indicating that MSC–PC direct contact or their presence in close vicinity plays a role in maintaining the functional viability of PCs. Fig. 5. View largeDownload slide The effect of PC–MSC separation on functional survival of PCs in vitro and co-localization of PCs with MSCs in vivo. (A) Determination of the CXCL12 concentration in the supernatants of cultured PCs with or without MSCs by ELISA. Values are expressed as means + SD for three independent experiments performed in duplicate. (B) Measurement of IgG and IgM titers in supernatants by ELISA. BM PCs were cultured with MSCs together or separated via transwells. Values are expressed as means + SD for three independent experiments. Statistical difference was analyzed by one-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) A minority of PCs are in proximity to MSCs in BM. Femoral BM sections from BlimpGFP mice were stained with fluorescently labeled anti-GFP antibody for PCs and PDGFRα and Sca-1 antibodies for MSCs. A representative picture is shown, and an image for the co-localization of a GFP+ cell and a PDGFRα+Sca-1+ cell is enlarged. Fig. 5. View largeDownload slide The effect of PC–MSC separation on functional survival of PCs in vitro and co-localization of PCs with MSCs in vivo. (A) Determination of the CXCL12 concentration in the supernatants of cultured PCs with or without MSCs by ELISA. Values are expressed as means + SD for three independent experiments performed in duplicate. (B) Measurement of IgG and IgM titers in supernatants by ELISA. BM PCs were cultured with MSCs together or separated via transwells. Values are expressed as means + SD for three independent experiments. Statistical difference was analyzed by one-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) A minority of PCs are in proximity to MSCs in BM. Femoral BM sections from BlimpGFP mice were stained with fluorescently labeled anti-GFP antibody for PCs and PDGFRα and Sca-1 antibodies for MSCs. A representative picture is shown, and an image for the co-localization of a GFP+ cell and a PDGFRα+Sca-1+ cell is enlarged. Given that MSCs supported PC function when they were co-cultured in vitro, we next wanted to examine whether they are indeed co-localized in BM. To this end, we stained femoral BM sections from BlimpGFP mice with anti-GFP antibody for detection of PCs and anti-PDGFRα and anti-Sca-1 antibodies for MSCs. As shown in Fig. 5(C), several but not majority of GFP+ cells were found to be apparently located near PDGFRα+Sca-1+ cells. In our experimental settings, 65 (23%) cells among 282 GFP+ were located within 10 µm distance of PDGFRα+Sca-1+ cells, although it might be overestimation due to the fact that some immunofluorescent signals for PDGFRα+Sca-1+ cells were not sufficiently clear, which could lead to the underestimation of those cells. These results suggested a co-localization of a minority of PCs with MSCs in BM. PDGFRα+Sca-1+ MSCs are a heterogenous population with different gene expression profiles To obtain further clues as to the MSC-associated molecules participating in MSC-mediated support of functional PC survival, we took advantage of a means of exhaustive analysis of transcripts, the scRNA-seq technique, in MSCs. Firstly, scRNA-seq of PDGFRα+Sca-1+ MSCs, and PDGFRα+Sca-1− and PDGFRα−Sca-1+ cells (PDGFRα+ cells and Sca-1+ cells, respectively) as controls (Fig. 6A) revealed that, at a false discovery rate (FDR) cut-off of 0.1, extracellular matrix components such as collagen and proteoglycan, and cytoskeleton-related factors such as actin and vimentin were abundantly expressed in PDGFRα+Sca-1+ MSCs (Supplementary Table S1). Next, we performed gene ontology (GO) analysis of the top 500 genes, which were significantly abundant in MSCs. GO analysis revealed that the extracellular domain, extracellular matrix and immune system response were significantly enriched in these genes (Fig. 6B). Then, we focused on the expression levels of the genes for PDGFRα+Sca-1+ MSC-sorting markers, Pdgfra and Ly6a (Sca-1). As expected, MSCs exhibited significantly higher expression of Pdgfra and Ly6a compared to PDGFRα+ cells and Sca-1+ cells (Fig. 6C, left). Gene expression of other MSC markers, Itgb1, Itga5, Cd44 and Thy1 (CD90) was significantly higher in MSCs (Fig. 6D, left), whereas Eng (CD105) expression was higher in Sca-1+ cells, which was consistent with the surface expression observed on flow cytometry analyzed at day 0 culture (Fig. 6D, left; Supplementary Figure S1). Overall, the gene expression analysis of sort-purified MSCs showed a similar profile to that on flow cytometric analysis, and the expression levels of the genes for stem cell markers were indeed high in our isolated MSC preparation. Fig. 6. View largeDownload slide Single-cell RNA-seq of PDGFRα+Sca-1+ MSCs. (A) Sort layout for single cell sorting. After dead cells had been excluded with PI, PDGFRα+Sca-1+ cells (MSCs), PDGFRα+Sca-1− cells (PDGFRα+) and PDGFRα−Sca-1+ (Sca-1+) cells were sorted in the gate of CD45–Ter119− cells. (B) GO analysis of MSCs and non-MSCs (PDGFRα+ cells and Sca-1+ cells) as to the top 500 significant genes on the FDR. (C, D, F–H) Comparison of transcript counts with each sorted cell type (left) or each population of MSCs (right). Statistical difference was analyzed by one-way ANOVA multiple comparison test. Values are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Principal component analysis of single-cell RNA-seq data for MSCs (red, n = 82), PDGFRα+ cells (black, n = 42) and Sca-1+ cells (blue, n = 40). These cells could be divided into four different populations (PC1–4). Fig. 6. View largeDownload slide Single-cell RNA-seq of PDGFRα+Sca-1+ MSCs. (A) Sort layout for single cell sorting. After dead cells had been excluded with PI, PDGFRα+Sca-1+ cells (MSCs), PDGFRα+Sca-1− cells (PDGFRα+) and PDGFRα−Sca-1+ (Sca-1+) cells were sorted in the gate of CD45–Ter119− cells. (B) GO analysis of MSCs and non-MSCs (PDGFRα+ cells and Sca-1+ cells) as to the top 500 significant genes on the FDR. (C, D, F–H) Comparison of transcript counts with each sorted cell type (left) or each population of MSCs (right). Statistical difference was analyzed by one-way ANOVA multiple comparison test. Values are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Principal component analysis of single-cell RNA-seq data for MSCs (red, n = 82), PDGFRα+ cells (black, n = 42) and Sca-1+ cells (blue, n = 40). These cells could be divided into four different populations (PC1–4). Next we performed principal component analysis (PCA) using transcriptional counts for PDGFRα+Sca-1+ MSCs, PDGFRα+ cells and Sca-1+ cells. We found that MSCs were widely distributed, and we arbitrarily subdivided them into four groups, PC1–PC4 (Fig. 6E, red symbols). The majority of both PDGFRα+ cells and Sca-1+ cells converged into PC4, while the minor ones did so into PC3 (Fig. 6E, black and blue symbols). Thus, MSCs seemed to be a heterogenous population in terms of the gene expression profiles, and the cells in PC4 were supposed to be closely related to both PDGFRα+ cells and Sca-1+ cells on PCA. To clarify the characteristics of MSCs tentatively categorized to each group, we compared the gene transcription counts among them. Pdgfra and Ly6a were the highest in PC2, and the lowest in PC4 (Fig. 6C, right). Other MSCs marker genes, Itgb1, Cd44 and Eng, were more highly expressed in PC1 than in the other groups, but Itga5 and Thy1 were less expressed in PC1 (Fig. 6D, right). On ELISA analysis, MSCs were found to secrete IL-6 and CXCL12 (Figs 3B and 5A). On PCA analysis, we found that the cells showing expression of mRNAs for them could be classified into different groups. Thus, the cells showing expression of Il6 mRNA could be classified into PC1, while PC3 and PC4 contained cells showing Cxcl12 expression (Fig. 6F and G, right). It is interesting to note that Vcam1 expression in MSCs was significantly high in the cells in PC1 (Fig. 6H, right), because IgG+ BM PCs were associated with CXCL12+VCAM1+ stromal cells (3). Counts for collagen-related genes such as Col1a1, Col1a2 and Col3a1, and Dcn (proteoglycan Decorin) were relatively high in PC2 (Supplementary Figure S3A), and the counts for genes coding for other extracellular matrix proteins, Fn1, Prg4 and Itga6, and Ccnd2 (cyclin CCND2) were high in PC1 (Supplementary Figure S3B and C). Although the gene expression profiles of PC3 and PC4 were grossly similar, that of Rn18s-rs5 (18S ribosomal protein-related sequence 5), which is important for protein synthesis (33), was markedly different (Supplementary Figure 3D, right). Also noteworthy is that expression of the Nestin protein-coding gene Nes was significantly high in PC1 (Supplementary Figure 3E, right), because Nestin+ MSCs form a niche for HSCs (22). In summary, the cells in the PC1 group expressed genes related to PC and HSC functions such as Vcam1, Nes and Il6, while those in both the PC1 and PC2 groups were grossly positive for gene expression of stem cell markers. On the other hand, the cells in PC3 and PC4 were characterized by roughly similar expression profiles of MSC markers as well as extracellular matrix components such as collagen, integrin and fibronectin. We concluded that our sort-purified PDGFRα+Sca-1+ MSCs supported BM PCs and were heterogenous in terms of the gene expression profiles. Discussion In this study, we investigated the in vitro effect of PDGFRα+Sca-1+ MSCs isolated from BM cells on the functional survival of BM PCs. We showed that the MSCs supported the survival of and antibody production by the PCs. We also showed that soluble factors including IL-6 played an important part in this support of PCs. While PC–BM stromal cell contact via a CD44 variant isoform was reported to induce IL-6 production by stromal cells (34), we failed to demonstrate a significant effect of the cell–cell contact on PC survival in this study. CD44 is involved in cell–cell and cell–extracellular matrix adhesion, and survival of IgG1-producing PCs was maintained on culture with anti-CD44 antibodies (10). CD44 is a hyaluronic acid receptor and interacts also with osteopontin, collagen and matrix metalloproteinases (MMPs) (35). CD44 itself is a surface marker of MSCs (14), and osteopontin and MMPs were detected in our culture supernatants on cytokine arraying. Although a whole spectrum of soluble factors and a potential cell–cell contact factor(s) in our PC–MSC culture system was not clarified, cell-adhesion molecules such as CD44 and related ligands could be involved in this system. Our scRNA-seq and PCA revealed that PDGFRα+Sca-1+ MSCs derived from BM comprise a heterogenous population, tentatively grouped into PC1–4 in terms of the gene expression profiles. Expression of Pdgfra and Ly6a was the highest in PC2 among them and it was also positive for other MSC markers. PC1 cells exhibited higher expression of Itgb1, Cd44 and Eng than PC2 ones did, whereas their expression of Itga5 and Thy1 was very low. Like PDGFRα+Sca-1− cells and PDGFRα−Sca-1+ cells, PC4 cells did not express stem cell markers, suggesting that they are going to undergo differentiation. PC3 cells seemed to have an intermittent gene expression profile between those of PC2 and PC4. On the basis of these observations, PC2 cells possessed most of the MSC profiles, while PC3 and PC4 cells were regarded as ones differentiating from MSCs. Then, the positioning of PC1 among these groups is obscure. The gene expression profiles of the extracellular matrix factors and others were different between PC1 and PC2. PC2 cells exhibited relatively high expression levels of collagen-related genes (Supplementary Figure S3A), while PC1 had high expression counts for Fn1, Prg4, Itga6 and Ccnd2 (Supplementary Figure S3B and C). These distinguishable profiles of PC1 and PC2 may be utilizable for further separation of these cell groups, which will enable us to clarify the positioning of PC1 cells. CXCL12+VCAM-1+ BM stromal cells, also known as CAR cells for CXCL12-abundant reticular cells, constitute a niche for BM PCs (3). It is widely accepted that the CXCL12–CXCR4 axis constituted primarily by CAR cells is important, but other CXCR4+ cells including eosinophils also cluster with the CXCL12+ stromal cells, thus forming a survival environment for PCs (4–9). These clustering cells secrete factors such as IL-6 and APRIL (10–12, 36). Because CAR cells are Sca-1− (37), our MSC preparation, which is Sca-1+, is different from CAR cells. Thus, our findings indicate that MSCs can also form a survival niche for BM PCs. On the other hand, our PCA study showed that VCAM-1+ cells and IL-6-producing cells are in PC1, but CXCL12-secreting cells in PC3. Although we do not have data regarding the spatial localization of these groups of MSCs in vivo, it is interesting to postulate that while CXCL12+ PC3 cells attract PCs in BM, IL-6-producing VCAM-1+ PC1 cells maintain their functional survival. Since IL-6 has also been shown to maintain the stemness of MSCs (38), PC1 cells may also contribute to maintenance of the stemness. Also, since Nestin+ MSCs were found in PC1 cells, it is suggested that HSCs and PCs are supported by PC1 cells together in BM. In this study, we showed a novel role of MSCs in BM for supporting the functional survival of PCs. Clarifying the molecular interactions between MSCs and BM PCs will provide us with an insight into the mechanism of long-term immunological memory as well as potential tools for regulating the memory. Funding Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15KK0328 and 17K15714 to A.I.-N. and 16H05201 and 17K19539 to T.T.), GSK Japan Research Grant 2015 and Naito Foundation Grant-in-Aid for Challenging Exploratory Research to A.I.-N., and Sumitomo Foundation to A.I.-N. and A.K. Conflicts of interest statement The authors declare no conflicts of interest. Acknowledgements We thank Mami Kikuchi, Makiko Nakagawa and Kiyotaka Kuroda (Tohoku University Graduate School of Medicine) for their technical assistance, and Nicholas Halewood for the editorial assistance. We also acknowledge the technical support of the Biomedical Research Core of Tohoku University Graduate School of Medicine. References 1 Ahmed , R. and Gray , D . 1996 . Immunological memory and protective immunity: understanding their relation . Science 272 : 54 . Google Scholar CrossRef Search ADS PubMed 2 Andraud , M. , Lejeune , O. , Musoro , J. Z. , Ogunjimi , B. , Beutels , P. and Hens , N . 2012 . Living on three time scales: the dynamics of plasma cell and antibody populations illustrated for hepatitis a virus . PLoS Comput. Biol . 8 : e1002418 . Google Scholar CrossRef Search ADS PubMed 3 Tokoyoda , K. , Egawa , T. , Sugiyama , T. , Choi , B. I. and Nagasawa , T . 2004 . Cellular niches controlling B lymphocyte behavior within bone marrow during development . Immunity 20 : 707 . Google Scholar CrossRef Search ADS PubMed 4 Chu , V. T. , Fröhlich , A. , Steinhauser , G. et al. 2011 . Eosinophils are required for the maintenance of plasma cells in the bone marrow . Nat. Immunol . 12 : 151 . Google Scholar CrossRef Search ADS PubMed 5 Rodriguez Gomez , M. , Talke , Y. , Goebel , N. , Hermann , F. , Reich , B. and Mack , M . 2010 . Basophils support the survival of plasma cells in mice . J. Immunol . 185 : 7180 . Google Scholar CrossRef Search ADS PubMed 6 Jinquan , T. , Jacobi , H. H. , Jing , C. et al. 2000 . Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4 . J. Allergy Clin. Immunol . 106 : 313 . Google Scholar CrossRef Search ADS PubMed 7 Winter , O. , Moser , K. , Mohr , E. et al. 2010 . Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow . Blood 116 : 1867 . Google Scholar CrossRef Search ADS PubMed 8 Rozanski , C. H. , Arens , R. , Carlson , L. M. et al. 2011 . Sustained antibody responses depend on CD28 function in bone marrow-resident plasma cells . J. Exp. Med . 208 : 1435 . Google Scholar CrossRef Search ADS PubMed 9 Glatman Zaretsky , A. , Konradt , C. , Dépis , F. et al. 2017 . T regulatory cells support plasma cell populations in the bone marrow . Cell Rep . 18 : 1906 . Google Scholar CrossRef Search ADS PubMed 10 Cassese , G. , Arce , S. , Hauser , A. E. et al. 2003 . Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals . J. Immunol . 171 : 1684 . Google Scholar CrossRef Search ADS PubMed 11 Benson , M. J. , Dillon , S. R. , Castigli , E. et al. 2008 . Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL . J. Immunol . 180 : 3655 . Google Scholar CrossRef Search ADS PubMed 12 Jourdan , M. , Cren , M. , Robert , N. et al. 2014 . IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors . Leukemia 28 : 1647 . Google Scholar CrossRef Search ADS PubMed 13 Minges Wols , H. A. , Underhill , G. H. , Kansas , G. S. and Witte , P. L . 2002 . The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity . J. Immunol . 169 : 4213 . Google Scholar CrossRef Search ADS PubMed 14 Houlihan , D. D. , Mabuchi , Y. , Morikawa , S. et al. 2012 . Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α . Nat. Protoc . 7 : 2103 . Google Scholar CrossRef Search ADS PubMed 15 Pittenger , M. F. , Mackay , A. M. , Beck , S. C. et al. 1999 . Multilineage potential of adult human mesenchymal stem cells . Science 284 : 143 . Google Scholar CrossRef Search ADS PubMed 16 Krampera , M. , Glennie , S. , Dyson , J. et al. 2003 . Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide . Blood 101 : 3722 . Google Scholar CrossRef Search ADS PubMed 17 Ren , G. , Zhang , L. , Zhao , X. et al. 2008 . Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide . Cell Stem Cell 2 : 141 . Google Scholar CrossRef Search ADS PubMed 18 Asari , S. , Itakura , S. , Ferreri , K. et al. 2009 . Mesenchymal stem cells suppress B-cell terminal differentiation . Exp. Hematol . 37 : 604 . Google Scholar CrossRef Search ADS PubMed 19 Schena , F. , Gambini , C. , Gregorio , A. et al. 2010 . Interferon-γ-dependent inhibition of B cell activation by bone marrow-derived mesenchymal stem cells in a murine model of systemic lupus erythematosus . Arthritis Rheum . 62 : 2776 . Google Scholar CrossRef Search ADS PubMed 20 Che , N. , Li , X. , Zhang , L. et al. 2014 . Impaired B cell inhibition by lupus bone marrow mesenchymal stem cells is caused by reduced CCL2 expression . J. Immunol . 193 : 5306 . Google Scholar CrossRef Search ADS PubMed 21 Luz-Crawford , P. , Djouad , F. , Toupet , K. et al. 2016 . Mesenchymal stem cell-derived interleukin 1 receptor antagonist promotes macrophage polarization and inhibits B cell differentiation . Stem Cells 34 : 483 . Google Scholar CrossRef Search ADS PubMed 22 Méndez-Ferrer , S. , Michurina , T. V. , Ferraro , F. et al. 2010 . Mesenchymal and haematopoietic stem cells form a unique bone marrow niche . Nature 466 : 829 . Google Scholar CrossRef Search ADS PubMed 23 El Haddad , N. , Heathcote , D. , Moore , R. et al. 2011 . Mesenchymal stem cells express serine protease inhibitor to evade the host immune response . Blood 117 : 1176 . Google Scholar CrossRef Search ADS PubMed 24 da Silva Meirelles , L. , Chagastelles , P. C. and Nardi , N. B . 2006 . Mesenchymal stem cells reside in virtually all post-natal organs and tissues . J. Cell Sci . 119 ( Pt 11 ): 2204 . Google Scholar CrossRef Search ADS PubMed 25 Phinney , D. G. , Kopen , G. , Isaacson , R. L. and Prockop , D. J . 1999 . Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation . J. Cell. Biochem . 72 : 570 . Google Scholar CrossRef Search ADS PubMed 26 Peister , A. , Mellad , J. A. , Larson , B. L. , Hall , B. M. , Gibson , L. F. and Prockop , D. J . 2004 . Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential . Blood 103 : 1662 . Google Scholar CrossRef Search ADS PubMed 27 Ohinata , Y. , Payer , B. , O’Carroll , D. et al. 2005 . Blimp1 is a critical determinant of the germ cell lineage in mice . Nature 436 : 207 . Google Scholar CrossRef Search ADS PubMed 28 Luckashenak , N. A. , Ryszkiewicz , R. L. , Ramsey , K. D. and Clements , J. L . 2006 . The Src homology 2 domain-containing leukocyte protein of 76-kDa adaptor links integrin ligation with p44/42 MAPK phosphorylation and podosome distribution in murine dendritic cells . J. Immunol . 177 : 5177 . Google Scholar CrossRef Search ADS PubMed 29 Hashimshony , T. , Senderovich , N. , Avital , G. et al. 2016 . CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq . Genome Biol . 17 : 77 . Google Scholar CrossRef Search ADS PubMed 30 Anderson , P. , Carrillo-Gálvez , A. B. , García-Pérez , A. , Cobo , M. and Martín , F . 2013 . CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities . PLoS One 8 : e76979 . Google Scholar CrossRef Search ADS PubMed 31 Stephan , R. P. , Reilly , C. R. and Witte , P. L . 1998 . Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age . Blood 91 : 75 . Google Scholar PubMed 32 Underhill , G. H. , Kolli , K. P. and Kansas , G. S . 2003 . Complexity within the plasma cell compartment of mice deficient in both E- and P-selectin: implications for plasma cell differentiation . Blood 102 : 4076 . Google Scholar CrossRef Search ADS PubMed 33 Rowe , L. B. , Janaswami , P. M. , Barter , M. E. and Birkenmeier , E. H . 1996 . Genetic mapping of 18S ribosomal RNA-related loci to mouse chromosomes 5, 6, 9, 12, 17, 18, 19, and X . Mamm. Genome 7 : 886 . Google Scholar CrossRef Search ADS PubMed 34 Van Driel , M. , Günthert , U. , van Kessel , A. C. et al. 2002 . CD44 variant isoforms are involved in plasma cell adhesion to bone marrow stromal cells . Leukemia 16 : 135 . Google Scholar CrossRef Search ADS PubMed 35 Ponta , H. , Sherman , L. and Herrlich , P. A . 2003 . CD44: from adhesion molecules to signalling regulators . Nat. Rev. Mol. Cell Biol . 4 : 33 . Google Scholar CrossRef Search ADS PubMed 36 Kometani , K. and Kurosaki , T . 2015 . Differentiation and maintenance of long-lived plasma cells . Curr. Opin. Immunol . 33 : 64 . Google Scholar CrossRef Search ADS PubMed 37 Sugiyama , T. and Nagasawa , T . 2012 . Bone marrow niches for hematopoietic stem cells and immune cells . Inflamm. Allergy Drug Targets 11 : 201 . Google Scholar CrossRef Search ADS PubMed 38 Pricola , K. L. , Kuhn , N. Z. , Haleem-Smith , H. , Song , Y. and Tuan , R. S . 2009 . Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism . J. Cell. Biochem . 108 : 577 . Google Scholar CrossRef Search ADS PubMed © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Immunology Oxford University Press

Bone marrow PDGFRα+Sca-1+-enriched mesenchymal stem cells support survival of and antibody production by plasma cells in vitro through IL-6

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Abstract

Abstract Plasma cells (PCs) acquiring long lifespans in the bone marrow (BM) play a pivotal role in the humoral arm of immunological memory. The PCs reside in a special BM niche and produce antibodies against past-encountered pathogens or vaccine components for a long time. In BM, cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4)-expressing PCs and myeloid cells such as dendritic cells are attracted to and held by CXC chemokine ligand 12 (CXCR12)-secreting stromal cells, where survival of the PCs is supported by soluble factors such as IL-6 and APRIL (a proliferation-inducing ligand) produced by neighboring myeloid cells. Although these stromal cells are also supposed to be involved in the support of the survival and antibody production, the full molecular mechanism has not been clarified yet. Here, we show that BM PDGFRα+Sca-1+-enriched mesenchymal stem cells (MSCs), which can contribute as stromal cells for hematopoietic stem cells, also support in vitro survival of and antibody production by BM PCs. IL-6 produced by MSCs was found to be involved in the support. Immunohistochemistry of BM sections suggested a co-localization of a minor population of PCs with PDGFRα+Sca-1+ MSCs in the BM. We also found that the sort-purified MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing, to which multiple roles in the in vitro PC support could be attributed. bone marrow niche, immunological memory, long-term antibody secretion Introduction Immunological memory is a highly integrated system composed of the coordinated functions of memory T and B cells as well as long-term antibody-secreting plasma cells (PCs) and the survival niches for those cells. Long-lived PCs maintain the humoral arm of the memory of previously experienced pathogens as well as vaccine components, so they provide effective secondary immune responses by having long lives in a special niche (1). It is important to clarify the molecular mechanism underlying the long life of PCs in a niche, because we do not have effective tools for regulating the magnitude and duration of antigen-specific antibody memory, which will be particularly important for eliminating harmful memories such as those established in allergies and autoimmune diseases. B cells can differentiate into two kinds of memory cells, namely memory B cells and PCs when stimulated by antigens and cytokines with cognate T-cell assistance. PCs then move to appropriate niches such as bone marrow (BM), acquire long-life ability and survive for several decades with continuous production of antibodies (2). The PCs in BM express cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4) and are located near the stromal cells expressing the CXCR4 ligand, CXC chemokine ligand 12 (CXCL12) and vascular cell adhesion molecule-1 (VCAM-1) (3). Other CXCR4+ cells such as eosinophils, basophils, megakaryocytes, dendritic cells and regulatory T cells also cluster with the CXCL12+ stromal cells, thus forming a survival environment for PCs (4–9). These clustering cells secrete factors contributing to the survival of and antibody production by PCs, such as IL-6, a proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), tumor necrosis factor α (TNFα) and CXCL12 (10–12). Co-culturing experiments on BM stromal cells and PCs in vitro also indicated that these cells maintain the longevity of PCs (13). These preceding studies indicated that various kinds of cells and molecules affect the survival and functions of long-lived PCs. However, the mechanisms as well as precise nature of the stromal cells have not been fully characterized yet. In BM, mesenchymal stem cells (MSCs) comprise a very small population accounting for only 0.05–0.08% of the total mononuclear cells (14). The MSCs in BM are multipotent cells able to differentiate into adipocytes, osteocytes and myocytes (15). On the other hand, MSCs in BM can have immunosuppressive functions, such as repression of proliferation of and cytokine production by T cells (16, 17), inhibition of activation and proliferation of B cells and their differentiation into PCs (18–21). In addition, Nestin+ MSCs form a niche for maintaining hematopoietic stem cells (HSCs) in BM (22). Although MSCs occupy only a small compartment in BM, we hypothesized that these cells might also play a role in the maintenance of PCs in BM, on the basis of the knowledge of their immunosuppressive functions as well as a role for HSCs. The conventional method for isolation of MSCs from BM cells is based on their adhesive nature as to plastic dishes (23). Since this isolation protocol takes several weeks, MSCs tend to differentiate into other types of cells (24). In addition, MSCs prepared by this protocol are heterozygous and possibly include HSCs and other adherent cells (25, 26). In order to test our hypothesis as to MSCs’ potential role in maintenance of BM PCs, we considered that choosing a suitable method for purifying MSCs was important. Houlihan et al. (14) established a means of flow cytometric isolation of MSCs using cell-surface MSC-specific markers, platelet-derived growth factor receptor alpha (PDGFRα) and stem cell antigen-1 (Sca-1), which enables us to obtain highly purified MSCs. By using this protocol, we expected that our hypothesis could be reasonably verified under conditions minimizing any effect of contaminating cells in the MSC preparation. In this study, we sort-purified PDGFRα+Sca-1+ MSCs from BM cells employing the flow cytometric protocol, co-cultured them with CD138+B220− PCs also isolated from BM cells by flow cytometry and evaluated the PC survival and antibody production by enzyme-linked immunosorbent spot (ELISpot) formation and enzyme-linked immunosorbent assaying (ELISA). We found that soluble factors including IL-6 contribute to the survival of and antibody production by PCs. We also showed that the isolated MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing (scRNA-seq), suggesting multiple roles are played by different groups of the MSCs. Methods Mice C57BL/6 (B6) mice were purchased from CLEA Japan (Shizuoka, Japan), and IL-6−⁄− mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The Blimp1-GFP transgenic mice (27) were kept in a B6 genetic background. Mice were maintained and bred in the animal facility of the Institute of Development, Aging and Cancer, Tohoku University, an environmentally controlled and specific pathogen-free facility, according to the guidelines for experimental animals defined by the university, and all animal experiments were approved by the Tohoku University Animal Studies Committee. All experiments were performed on 6- to 32-week-old male and female mice. Antibodies and flow cytometry Allophycocyanin (APC)-labeled rat anti-mouse PDGFRα (Clone APA5), APC-labeled rat anti-mouse CD138 (281-2), FITC-labeled rat anti-mouse Sca-1 (D7), PE-labeled rat anti-mouse CD45 (30-F11), PE-labeled Armenian hamster anti-mouse/rat CD29 (HMβ1-1), PE-labeled Armenian hamster anti-mouse/rat CD49e (HMα5-1), PE-labeled rat anti-mouse CD90.2 (53-2.1), PE-labeled rat anti-mouse CD105 (MJ7/18) and PE-labeled rat anti-mouse TER-119 (TER-119) were purchased from BioLegend (San Diego, CA, USA). PE-labeled rat anti-mouse CD44 (IM7) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Cells were incubated with staining reagents in staining buffer [1% bovine serum albumin (BSA) in PBS] for 30 min at 4°C. Cells were acquired with a FACS Aria III using FACS Diva software (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Cell isolation by flow cytometry PDGFRα+Sca-1+CD45−TER-119− MSCs were isolated from femoral and tibial BM of 6–10-week-old mice with a FACS Aria III (BD Biosciences) according to the established method (14). CD138+B220− PCs were isolated from femoral and tibial BM of 26–32-week-old mice by flow cytometry. Cell culture Isolated MSCs were suspended at a density of 3 × 104 cells ml−1, and a 100 µl aliquot was applied to each well of 96-well plates. Three days later, sort-purified PCs suspended at 8 × 104 cells ml−1 were seeded at 100 µl per well for the MSC culture. The culture was conducted in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) containing penicillin/streptomycin (Sigma-Aldrich), 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 10 mM HEPES (Dojindo Laboratories, Kumamoto, Japan), 1 mM sodium pyruvate (Sigma-Aldrich), 0.1 mM MEM-NEAA (Thermo Fisher Scientific) and 50 µM 2-mercaptoethanol (Sigma-Aldrich) in a humidified atmosphere of 95% air and 5% CO2 at 37°C for the indicated period of time. For assessing a role of IL-6, recombinant mouse IL-6 (Wako Pure Chemical Industries, Osaka, Japan) and/or LEAF purified anti-mouse IL-6 antibody (BioLegend) were added to culture as indicated. Generation of BM-derived cultured dendritic cells BM cells prepared from femoral and tibial BM of B6 mice were cultured with 20 ng ml−1 GM-CSF (Peprotech, Rocky Hill, NJ, USA) for 7 days (28) to obtain BM-derived cultured dendritic cells (BMDCs). At day 6, lipopolysaccharide (Sigma-Aldrich) was added at 1 µg ml−1 to promote maturation. ELISpot assaying For detection of IgG-secreting cells, Elispot MultiScreenHTS Filter Plates (Merck Millipore, Billerica, MA, USA) were coated with 10 µg ml−1 affinity-purified goat anti-mouse IgG-Fc antibodies (Bethyl Laboratories) and then incubated overnight at 4°C. The plates were blocked with RPMI culture medium for 90 min. Freshly sort-purified or cultured PCs were added at a volume of 200 µl per well, followed by incubation at 37°C. After 4-h incubation, the plates were treated with HRP-conjugated goat anti-mouse IgG-Fc detection antibodies (Bethyl Laboratories) and developed with 3-amino-9-ethylcarbazole. Spots were examined under an upright microscope (BX53, Olympus, Tokyo, Japan) and analyzed with ImageJ 1.42q software. The cell survival rate was calculated from the spot number after day 7 or day 14 culture compared with that on day 0. Microscopic analysis Images of immunofluorescently labeled cells were acquired on a fluorescence microscope BZ-X700 (Keyence, Osaka, Japan), and analyzed with BZ-X analyzer (Keyence) and ImageJ software. Numbers of GFP+ PCs and propidium iodide (PI)+ PCs were counted automatically by BZ-X analyzer (Keyence). After automated enumeration of PI+ cells with the BZ-X analyzer, we excluded the counts for the cells larger than 400 µm2, which was the maximum area for PCs observed in the culture without MSCs. Then, the cells having apparently abnormal shapes being different from that of typical PCs were visually excluded. Immunohistochemical analysis Mouse femurs were isolated and frozen BM sections were prepared according to the Kawamoto’s film method. Frozen blocks were cut into 5-µm sections, using a Microm HM505E cryostat, and fixed in 4% paraformaldehyde for 10 min. After blocking with 2% BSA–PBS with 5% rat serum (Wako) for 1 h, samples were incubated at 4°C overnight with biotin-labeled anti-PDGFRα antibody (1:40, BioLegend), Alexa Fluor 647 anti-Sca-1 antibody (1:50, BioLegend), GFP-tag polyclonal antibodies (1:1000, Thermo Fisher Scientific) in 1% BSA–PBS. The secondary antibody reaction for biotinylated PDGFRα was streptavidin Alexa Fluor 594 antibody (1:500, Invitrogen), and for GFP was Alexa Fluor 488 anti-rabbit IgG antibody (1:1000, Invitrogen) at room temperature for 1 h. ELISA Culture supernatants were frozen at −80°C until analyzed by ELISA. For detection of total IgG and IgM secreted by PCs, mouse IgG and IgM ELISA Quantitation Sets (Bethyl Laboratories) were used. For detection of IL-6, a Mouse IL-6 ELISA MAX™ Standard Set (BioLegend) was used. For detection of CXCL12, Quantikine ELISA Mouse CXCL12/SDF-1α (R&D Systems) was used. Each assay was performed according to the manufacturer’s instructions. Real-time PCR for quantitation of IL-6 mRNA RNA was isolated using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized using a ReverTra Ace PCR RT Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The cDNA was analyzed by quantitative PCR with SYBR Green Realtime PCR Master Mix Plus (Toyobo) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Primer sequences for Il6 were: forward, 5′-AGCCAGAGTCCTTCAGAGAGAT-3′; reverse, 5′-GAGAGCATTGGAAATTGGGGT-3′; for actb were: 5′-CGTTGACATCCGTAAAGACCTC-3′; reverse, 5′-AGCCACC GATCCACACAGA-3′. Transwell assaying Transwell cultures were set up in 24-well plates. ThinCert™ cell culture inserts with 0.4 µm-pore membranes of 1 × 108 cm−2 pore density (Greiner Bio-One, Kremsmunster, Austria) were used to physically separate PCs from MSCs. Plasma cells (8 × 103 cells per well) were cultured in the upper chamber, and MSCs (1 × 104 cells per well) in the lower chamber. Cytokine array A Mouse XL Cytokine Array Kit (R&D Systems) consisting of a total 111 different cytokine and chemokine antibodies spotted in duplicate onto the membrane was used according to the manufacturer’s directions. The membranes were developed with an ImageQuant LAS 4000 mini (GE Healthcare Japan, Tokyo, Japan) and analyzed with an ImageQuant TL (GE Healthcare Japan). Single-cell RNA sequencing Single-cell RNA sequencing (scRNA-seq) was performed with multiplex linear amplification2 (CEL-Seq2) as described previously (29). An MSC was sorted into a well containing 1.2 µl primer mix in a 96-well plate. After cell disruption at 65°C for 5 min, reverse transcription was performed at 42°C for 1 h with 0.4 µl first strand buffer, 0.2 µl 0.1 M DTT, 0.1 µl RNase Inhibitor and 0.1 µl Superscript II. The reverse transcript was inactivated by heating the mixture at 70°C for 10 min. The second strand reaction was performed at 16°C for 2 h with 7 µl H2O, 2.31 µl second strand buffer, 0.23 µl dNTP, ligase 0.08 µl, Escherichia coli DNA polymerase and 0.08 µl RNaseH. Double-strand DNA (dsDNA) samples in a 96-well plate were collected and cDNA cleanup was performed by the method described previously (29). In vitro transcription was performed at 37°C for 13 h with 3.2 µl ATP, 3.2 µl GTP, 3.2 µl CTP, 3.2 µl UTP, 3.2 µl 10 × T7 buffer, 3.2 µl T7 enzyme and 12 µl dsDNA. RNA samples were subjected to EXO-SAP treatment, RNA fragmentation, RNA cleanup and library preparation for Illumina RNA-sequencing as previously described (29). The libraries were sequenced on HiSeq2500, controlled by HiSeq Control Software v2.2.58, with 15 bases for read 1 and 36 bases for read 2. Gene expression analysis Expression analysis was performed using the CEL-Seq-2 pipeline (https://github.com/yanailab/CEL-Seq-pipeline) (29). Briefly, paired-end fastq files for each RNA-seq sample were demultiplexed to generate 96 single-end fastq files, each of which represents expression from a single cell. The single-end reads were of 35 bases and attached to 5-base unique molecular identifiers (UMI). The reads were mapped on the mouse GRCm38 reference genome using the Bowtie2 program. Gene expression was quantified with the SAM files as the number of reads mapped on the exonic region of each gene according to the RefSeq gene annotation on GRCm38. UMI was used to remove the PCR duplication biases. Statistical analysis Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA) on the basis of the results of at least three independent experiments. Significance was assessed by the unpaired two-tailed Student’s t-test, one-way analysis of variance (ANOVA) or two-way ANOVA. P <0.05 was considered statistically significant. Results MSCs support the survival of and antibody production by BM PCs To investigate the effect of MSCs on the survival of and antibody production by PCs, we first isolated CD45−TER-119−PDGFRα+Sca-1+ MSCs (14) from BM cells of wild-type C57BL/6 mice using a flow cytometer and then cultured them in vitro without PCs (Fig. 1A). Twenty-two days later, we characterized the surface markers on cultured MSCs, and found that the MSCs maintained the expression of PDGFRα and Sca-1 (Fig. 1B). The expression of other MSC markers, CD44, CD29, CD90 and CD49e, was also positive, while CD105 expression was bi-phasic, the CD105low population comprising the majority (Fig. 1C). On BM stromal cells, it was shown that the expression of CD105 decreased depending on the passage and cell density (30), suggesting that also on BM MSCs the CD105 expression decreased during the culture. To examine this possibility, we measured the CD105 expression on MSCs on the first day of isolation, and found that it was substantially positive on the freshly isolated MSCs (Supplementary Figure S1). Overall, we concluded that the isolated MSCs had a similar expression profile of surface markers to that reported previously (14). Fig. 1. View largeDownload slide Isolation of MSCs and PCs among BM cells by flow cytometry. (A) Flow cytometric isolation of MSCs. After exclusion of dead cells by PI staining, CD45−TER-119−PDGFRα+Sca-1+ cells were gated and sort purified as MSCs. (B, C) Surface marker expression by MSCs at day 22 of culture (solid line). Dotted line indicates isotype control. (D) Flow cytometric isolation of PCs among BM cells. CD138+B220− cells were sort purified after exclusion of dead cells by PI staining. Fig. 1. View largeDownload slide Isolation of MSCs and PCs among BM cells by flow cytometry. (A) Flow cytometric isolation of MSCs. After exclusion of dead cells by PI staining, CD45−TER-119−PDGFRα+Sca-1+ cells were gated and sort purified as MSCs. (B, C) Surface marker expression by MSCs at day 22 of culture (solid line). Dotted line indicates isotype control. (D) Flow cytometric isolation of PCs among BM cells. CD138+B220− cells were sort purified after exclusion of dead cells by PI staining. Next, we sort-purified CD138+B220− PCs from BM cells (Fig. 1D) and plated them onto a day 3 MSC culture. We employed BMDCs and BM stromal cell line OP9 as positive controls, because BMDCs and BM stromal cells were shown to support the survival of and antibody secretion by BM PCs (8, 13, 31). BM PCs were also cultured alone as a negative control. After 7 days of co-culture, the culture supernatant was collected and subjected to measurement of IgG and IgM by ELISA. Also, PCs were recovered from the co-culture, the IgG-secreting cells among them being enumerated by ELISpot assaying, and then positive cell numbers were compared with those beforehand on day 0 as a measure of the survival rate of antibody-secreting cells. We found that the number of functionally surviving, antibody-secreting PCs was two to three times higher in the MSC co-culture than among PCs cultured alone as assessed by ELISpot (Fig. 2A, left graph). We also measured the individual spot areas as a measure for the antibody secretion activity of PCs and found that it was significantly increased in the MSC co-culture (Fig. 2A, right graph), suggesting that the activity was higher in the PCs under co-culture than those cultured alone. Fig. 2. View largeDownload slide Effect of co-culture of BM PCs with MSCs on their survival and antibody production. (A) Quantification of functionally surviving cells by counting IgG-secreting PCs by means of ELISpot after culturing with (right picture) or without (left picture) MSCs for 7 days. Survival rate % (left graph) is calculated by comparison to the ELISpot count at day 0 culture, and spot area (right graph) is measured by ImageJ. Values are expressed as the mean + SD for four independent experiments performed in duplicate. (B) Estimation of survival of PCs in culture under a fluorescence microscope. Representative fields are shown. Live PCs cultured alone or with MSCs for 7 days were visualized as to GFP expression. Original magnification, ×20. Each signal of GFP+ PCs and PI-stained cells was counted, and live cell % was calculated as follows: GFP+ cell number ⁄ sum of GFP+ and PI+ cell number × 100. Values are expressed as the means + SD of three independent experiments. (C) Determination of IgG (left) and IgM (right) concentrations by ELISA in day 7 culture supernatants of PCs co-cultured with or without MSCs, OP9 or BMDCs. The data are shown as fold changes as to PCs without co-culture (designated as 1). Values are expressed as means + SD for three independent experiments performed in duplicate. (D, E) Time course of the survival rate (D), and IgG and IgM levels (E) in culture supernatants of PCs co-cultured with or without MSCs for up to day 14. Values are expressed as means ± SD for three or four independent experiments performed in duplicate. Statistical differences were analyzed by means of the one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. 2. View largeDownload slide Effect of co-culture of BM PCs with MSCs on their survival and antibody production. (A) Quantification of functionally surviving cells by counting IgG-secreting PCs by means of ELISpot after culturing with (right picture) or without (left picture) MSCs for 7 days. Survival rate % (left graph) is calculated by comparison to the ELISpot count at day 0 culture, and spot area (right graph) is measured by ImageJ. Values are expressed as the mean + SD for four independent experiments performed in duplicate. (B) Estimation of survival of PCs in culture under a fluorescence microscope. Representative fields are shown. Live PCs cultured alone or with MSCs for 7 days were visualized as to GFP expression. Original magnification, ×20. Each signal of GFP+ PCs and PI-stained cells was counted, and live cell % was calculated as follows: GFP+ cell number ⁄ sum of GFP+ and PI+ cell number × 100. Values are expressed as the means + SD of three independent experiments. (C) Determination of IgG (left) and IgM (right) concentrations by ELISA in day 7 culture supernatants of PCs co-cultured with or without MSCs, OP9 or BMDCs. The data are shown as fold changes as to PCs without co-culture (designated as 1). Values are expressed as means + SD for three independent experiments performed in duplicate. (D, E) Time course of the survival rate (D), and IgG and IgM levels (E) in culture supernatants of PCs co-cultured with or without MSCs for up to day 14. Values are expressed as means ± SD for three or four independent experiments performed in duplicate. Statistical differences were analyzed by means of the one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Because recovery of the cultured PCs might not be quantitative, particularly in a co-culture with MSCs because of potential adherence to MSCs as well as to the culture plates, we also checked the survival of PCs in culture using a fluorescent microscopic technique. To this end, live PCs were visualized as their GFP expression driven by a Blimp1 promoter prepared from BM cells of BlimpGFP mice (27), while dead PCs were stained with PI, albeit PI+ MSCs were scarcely detected in our analysis. We enumerated live GFP+ PCs and dead PI+ PCs, calculated the PC survival % as the rate of GFP+ cells among total GFP+ plus PI+ cells, and found that the PC survival % was higher at day 7 co-culture with MSCs than that without MSCs (Fig. 2B), indicating that MSCs support the survival of PCs. On ELISA measurement, both the IgG and IgM levels in the culture supernatant were significantly higher in the PC–MSC co-culture than with PCs alone (Fig. 2C). PC co-culture with OP9 or BMDCs had a similar but not superior effect on the immunoglobulin production to that obtained for the co-culture with MSCs (Fig. 2C). To test whether this supportive effect of MSCs on maintenance of functional PCs observed after day 7 culture could be sustained for a longer period of time, the PC–MSC co-culture was extended up to day 14. We found that the functionally surviving cell rate observed at day 7 was still maintained at day 14. Likewise, the IgG and IgM levels in the culture supernatant were even higher at day 14 than those at day 7, indicating that functionally surviving PCs were mostly maintained from day 7 to day 14 (Fig. 2D and E). Collectively, these data indicated that MSCs supported the survival of and antibody production by BM PCs as efficiently as OP9 cells and BMDCs do. IL-6 produced by MSCs is a supporting factor for functional PC survival To elucidate the mechanism by which MSCs support the survival of and antibody production by PCs, we examined potential IL-6 secretion by MSCs, because IL-6 is known to support PC survival (10). Firstly, we measured expression of IL-6 mRNA by RT–PCR. We isolated MSCs, PDGFRα+Sca-1– (PDGFRα+) and PDGFRα–Sca-1+ (Sca-1+) cells and cultured for 7 days with or without PCs. IL-6 mRNA levels in MSCs were significantly higher than PDGFRα+ cells and Sca-1+ cells (Fig. 3A). In BM stromal cells, expression of IL-6 mRNA was shown to be up-regulated upon culture with PCs (13), suggesting a potential signal from PCs to MSCs that leads to IL-6 mRNA transcription. In our analysis, however, IL-6 mRNA was not induced by co-culture with PCs. Therefore, MSCs constitutively express IL-6 mRNA, which could be a unique characteristics of MSCs. Next, we measured the IL-6 concentration in the supernatant of BM PCs co-cultured with MSCs, OP9 or BMDCs for 7 days and found that MSCs and BMDCs but not PCs or OP9 secreted IL-6 (Fig. 3B, left). The IL-6 level in the supernatant was comparable to those between day 7 and day 14 PC–MSC co-culture (Fig. 3B, right). These results suggested that IL-6 could be a supporting factor for PC survival provided by MSCs and BMDCs in our culture system, albeit that the mechanism for PC support by OP9 would not be dependent on IL-6. To verify the roles of IL-6 and other unidentified factors in functional PC survival, we examined the effect of addition of mouse recombinant IL-6 (rIL-6) and neutralization of MSC-derived IL-6 by anti-IL-6 antibody on the functional survival of PCs. IgG levels were increased but they did not exceed the levels of MSC co-culture (Fig. 3C), suggesting an involvement of other stimulating factor(s) in MSC co-culture. In the experiment of neutralization of IL-6 by anti-IL-6 antibody, IgG levels were decreased compared to the positive control MSC co-culture, but still higher than PC culture (Fig. 3C). These results suggest that IL-6 plays a role in supporting PCs, while other factors are also involved in the support. In addition, we co-cultured PCs with MSCs isolated from BM cells of Il6-deficient mice. ELISpot analysis revealed that the survival rate was reduced, and the IgG and IgM concentrations in the supernatant were markedly reduced but not completely abolished in the co-culture with IL-6-deficient MSCs (Fig. 3D). These results suggested that IL-6 is a substantial supporting cytokine for functional PC survival but not an exclusive factor. Fig. 3. View largeDownload slide Reduced functional survival of PCs co-cultured with IL-6-deficient MSCs. (A) Quantitative real-time PCR to determine the levels of IL-6 mRNA. MSCs, PDGFRα+Sca-1– (PDGFRα+) cells and PDGFRα–Sca-1+ (Sca-1+) cells were cultured for 7 days with or without PCs. (B) IL-6 concentrations in culture supernatants of PCs with or without co-culture at day 7 (left), and changes of the IL-6 levels in the supernatants of PCs up to day 14 (right). Values are expressed as means ± SD for three independent experiments. (C) IgG (left), IgM (middle) and IL-6 (right) levels in supernatants of PCs cultured alone and with MSCs treated with recombinant IL-6 (rIL-6, 10 ng ml−1) or anti-IL-6 antibody (αIL-6, 10 µg ml−1). Ctrl. means with or without isotype control. (D) Functional survival rate (upper left) of PCs and IL-6 (upper right), IgG (lower left) and IgM (lower right) levels in supernatants of PCs, cultured alone or co-cultured with MSCs prepared from wild-type or Il6-deficient mice. Values are expressed as means + SD of three independent experiments performed in duplicate or triplicate. Statistical difference was analyzed by one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. 3. View largeDownload slide Reduced functional survival of PCs co-cultured with IL-6-deficient MSCs. (A) Quantitative real-time PCR to determine the levels of IL-6 mRNA. MSCs, PDGFRα+Sca-1– (PDGFRα+) cells and PDGFRα–Sca-1+ (Sca-1+) cells were cultured for 7 days with or without PCs. (B) IL-6 concentrations in culture supernatants of PCs with or without co-culture at day 7 (left), and changes of the IL-6 levels in the supernatants of PCs up to day 14 (right). Values are expressed as means ± SD for three independent experiments. (C) IgG (left), IgM (middle) and IL-6 (right) levels in supernatants of PCs cultured alone and with MSCs treated with recombinant IL-6 (rIL-6, 10 ng ml−1) or anti-IL-6 antibody (αIL-6, 10 µg ml−1). Ctrl. means with or without isotype control. (D) Functional survival rate (upper left) of PCs and IL-6 (upper right), IgG (lower left) and IgM (lower right) levels in supernatants of PCs, cultured alone or co-cultured with MSCs prepared from wild-type or Il6-deficient mice. Values are expressed as means + SD of three independent experiments performed in duplicate or triplicate. Statistical difference was analyzed by one-way ANOVA and two-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MSCs secrete a number of soluble factors To explore the soluble factors other than IL-6 supporting the survival of and antibody production by PCs, we employed a cytokine array technique for identifying cytokines, chemokines, and growth factors in culture supernatants of MSCs and ones of OP9 for comparison (Fig. 4A and B). We identified CCL2, CHI3L1, IGFBP-2, -5 and -6, M-CSF, MMP3 and Pentraxin 3 as the soluble factors secreted specifically by MSCs but not OP9 (Fig. 4C). IL-6, detected on ELISA (Fig. 3B) and also as its mRNA expression (Fig. 3A), did not give a significant signal with the cytokine array technique (Fig. 4A), potentially because of an insufficient sensitivity of the antibody used in this array. To determine whether these MSC-specific factors have a supportive effect on PCs, we added some recombinant proteins to the PC cultures without MSCs. However, while IL-6, as a positive control, markedly sustained the IgG and IgM levels, the others did not (Supplementary Figure S2). Other soluble factors commonly detected in both MSCs and OP9, such as CXCL1, Cystain C, LIX, Osteopontin and Serpin E1, might also be candidate(s) for MSC-mediated PC support, which awaits further investigation. Fig. 4. View largeDownload slide Profiles of soluble factors in culture supernatants of MSCs and OP9 cells. (A, B) Detection of cytokines, chemokines and growth factors in culture supernatants of MSCs and OP9 by cytokine array analysis, in which the supernatants were blotted onto membranes in duplicate (A), followed by detection with each of the specific antibodies shown in B. (C) Quantification of signal intensities shown in A by measuring pixel density. The factors in boldface are those exhibiting notably higher expression in MSCs than OP9. The factors with asterisks are those tested for the supporting effect by the inclusion to PC culture shown in Supplementary Figure S2. Fig. 4. View largeDownload slide Profiles of soluble factors in culture supernatants of MSCs and OP9 cells. (A, B) Detection of cytokines, chemokines and growth factors in culture supernatants of MSCs and OP9 by cytokine array analysis, in which the supernatants were blotted onto membranes in duplicate (A), followed by detection with each of the specific antibodies shown in B. (C) Quantification of signal intensities shown in A by measuring pixel density. The factors in boldface are those exhibiting notably higher expression in MSCs than OP9. The factors with asterisks are those tested for the supporting effect by the inclusion to PC culture shown in Supplementary Figure S2. Examination of a role of MSC–PC direct contact in supporting PCs BM PCs express CXCR4 and are located near CXCL12+ stromal cells (3, 32). To determine whether MSCs express CXCL12, we performed ELISA assaying. CXCL12 was detected in the supernatant of the MSC–PC co-culture, but it was negligible in the PC culture without MSCs (Fig. 5A), suggesting a potential of MSCs for producing CXCL12, secreting it into the milieu, attracting PCs and interacting with those in close vicinity in BM. Therefore, next we were interested in examining a potential role of MSC–PC direct contact in supporting PCs. To this end, we employed a transwell culture where BM PCs were placed in the upper chamber separated from MSCs in the bottom chamber by a semi-permeable membrane, and measured the IgG and IgM levels in the culture supernatant after 7 days. We found that the IgG and IgM levels in the culture supernatant were decreased upon separation (Fig. 5B), indicating that MSC–PC direct contact or their presence in close vicinity plays a role in maintaining the functional viability of PCs. Fig. 5. View largeDownload slide The effect of PC–MSC separation on functional survival of PCs in vitro and co-localization of PCs with MSCs in vivo. (A) Determination of the CXCL12 concentration in the supernatants of cultured PCs with or without MSCs by ELISA. Values are expressed as means + SD for three independent experiments performed in duplicate. (B) Measurement of IgG and IgM titers in supernatants by ELISA. BM PCs were cultured with MSCs together or separated via transwells. Values are expressed as means + SD for three independent experiments. Statistical difference was analyzed by one-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) A minority of PCs are in proximity to MSCs in BM. Femoral BM sections from BlimpGFP mice were stained with fluorescently labeled anti-GFP antibody for PCs and PDGFRα and Sca-1 antibodies for MSCs. A representative picture is shown, and an image for the co-localization of a GFP+ cell and a PDGFRα+Sca-1+ cell is enlarged. Fig. 5. View largeDownload slide The effect of PC–MSC separation on functional survival of PCs in vitro and co-localization of PCs with MSCs in vivo. (A) Determination of the CXCL12 concentration in the supernatants of cultured PCs with or without MSCs by ELISA. Values are expressed as means + SD for three independent experiments performed in duplicate. (B) Measurement of IgG and IgM titers in supernatants by ELISA. BM PCs were cultured with MSCs together or separated via transwells. Values are expressed as means + SD for three independent experiments. Statistical difference was analyzed by one-way ANOVA multiple comparison test and Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) A minority of PCs are in proximity to MSCs in BM. Femoral BM sections from BlimpGFP mice were stained with fluorescently labeled anti-GFP antibody for PCs and PDGFRα and Sca-1 antibodies for MSCs. A representative picture is shown, and an image for the co-localization of a GFP+ cell and a PDGFRα+Sca-1+ cell is enlarged. Given that MSCs supported PC function when they were co-cultured in vitro, we next wanted to examine whether they are indeed co-localized in BM. To this end, we stained femoral BM sections from BlimpGFP mice with anti-GFP antibody for detection of PCs and anti-PDGFRα and anti-Sca-1 antibodies for MSCs. As shown in Fig. 5(C), several but not majority of GFP+ cells were found to be apparently located near PDGFRα+Sca-1+ cells. In our experimental settings, 65 (23%) cells among 282 GFP+ were located within 10 µm distance of PDGFRα+Sca-1+ cells, although it might be overestimation due to the fact that some immunofluorescent signals for PDGFRα+Sca-1+ cells were not sufficiently clear, which could lead to the underestimation of those cells. These results suggested a co-localization of a minority of PCs with MSCs in BM. PDGFRα+Sca-1+ MSCs are a heterogenous population with different gene expression profiles To obtain further clues as to the MSC-associated molecules participating in MSC-mediated support of functional PC survival, we took advantage of a means of exhaustive analysis of transcripts, the scRNA-seq technique, in MSCs. Firstly, scRNA-seq of PDGFRα+Sca-1+ MSCs, and PDGFRα+Sca-1− and PDGFRα−Sca-1+ cells (PDGFRα+ cells and Sca-1+ cells, respectively) as controls (Fig. 6A) revealed that, at a false discovery rate (FDR) cut-off of 0.1, extracellular matrix components such as collagen and proteoglycan, and cytoskeleton-related factors such as actin and vimentin were abundantly expressed in PDGFRα+Sca-1+ MSCs (Supplementary Table S1). Next, we performed gene ontology (GO) analysis of the top 500 genes, which were significantly abundant in MSCs. GO analysis revealed that the extracellular domain, extracellular matrix and immune system response were significantly enriched in these genes (Fig. 6B). Then, we focused on the expression levels of the genes for PDGFRα+Sca-1+ MSC-sorting markers, Pdgfra and Ly6a (Sca-1). As expected, MSCs exhibited significantly higher expression of Pdgfra and Ly6a compared to PDGFRα+ cells and Sca-1+ cells (Fig. 6C, left). Gene expression of other MSC markers, Itgb1, Itga5, Cd44 and Thy1 (CD90) was significantly higher in MSCs (Fig. 6D, left), whereas Eng (CD105) expression was higher in Sca-1+ cells, which was consistent with the surface expression observed on flow cytometry analyzed at day 0 culture (Fig. 6D, left; Supplementary Figure S1). Overall, the gene expression analysis of sort-purified MSCs showed a similar profile to that on flow cytometric analysis, and the expression levels of the genes for stem cell markers were indeed high in our isolated MSC preparation. Fig. 6. View largeDownload slide Single-cell RNA-seq of PDGFRα+Sca-1+ MSCs. (A) Sort layout for single cell sorting. After dead cells had been excluded with PI, PDGFRα+Sca-1+ cells (MSCs), PDGFRα+Sca-1− cells (PDGFRα+) and PDGFRα−Sca-1+ (Sca-1+) cells were sorted in the gate of CD45–Ter119− cells. (B) GO analysis of MSCs and non-MSCs (PDGFRα+ cells and Sca-1+ cells) as to the top 500 significant genes on the FDR. (C, D, F–H) Comparison of transcript counts with each sorted cell type (left) or each population of MSCs (right). Statistical difference was analyzed by one-way ANOVA multiple comparison test. Values are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Principal component analysis of single-cell RNA-seq data for MSCs (red, n = 82), PDGFRα+ cells (black, n = 42) and Sca-1+ cells (blue, n = 40). These cells could be divided into four different populations (PC1–4). Fig. 6. View largeDownload slide Single-cell RNA-seq of PDGFRα+Sca-1+ MSCs. (A) Sort layout for single cell sorting. After dead cells had been excluded with PI, PDGFRα+Sca-1+ cells (MSCs), PDGFRα+Sca-1− cells (PDGFRα+) and PDGFRα−Sca-1+ (Sca-1+) cells were sorted in the gate of CD45–Ter119− cells. (B) GO analysis of MSCs and non-MSCs (PDGFRα+ cells and Sca-1+ cells) as to the top 500 significant genes on the FDR. (C, D, F–H) Comparison of transcript counts with each sorted cell type (left) or each population of MSCs (right). Statistical difference was analyzed by one-way ANOVA multiple comparison test. Values are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Principal component analysis of single-cell RNA-seq data for MSCs (red, n = 82), PDGFRα+ cells (black, n = 42) and Sca-1+ cells (blue, n = 40). These cells could be divided into four different populations (PC1–4). Next we performed principal component analysis (PCA) using transcriptional counts for PDGFRα+Sca-1+ MSCs, PDGFRα+ cells and Sca-1+ cells. We found that MSCs were widely distributed, and we arbitrarily subdivided them into four groups, PC1–PC4 (Fig. 6E, red symbols). The majority of both PDGFRα+ cells and Sca-1+ cells converged into PC4, while the minor ones did so into PC3 (Fig. 6E, black and blue symbols). Thus, MSCs seemed to be a heterogenous population in terms of the gene expression profiles, and the cells in PC4 were supposed to be closely related to both PDGFRα+ cells and Sca-1+ cells on PCA. To clarify the characteristics of MSCs tentatively categorized to each group, we compared the gene transcription counts among them. Pdgfra and Ly6a were the highest in PC2, and the lowest in PC4 (Fig. 6C, right). Other MSCs marker genes, Itgb1, Cd44 and Eng, were more highly expressed in PC1 than in the other groups, but Itga5 and Thy1 were less expressed in PC1 (Fig. 6D, right). On ELISA analysis, MSCs were found to secrete IL-6 and CXCL12 (Figs 3B and 5A). On PCA analysis, we found that the cells showing expression of mRNAs for them could be classified into different groups. Thus, the cells showing expression of Il6 mRNA could be classified into PC1, while PC3 and PC4 contained cells showing Cxcl12 expression (Fig. 6F and G, right). It is interesting to note that Vcam1 expression in MSCs was significantly high in the cells in PC1 (Fig. 6H, right), because IgG+ BM PCs were associated with CXCL12+VCAM1+ stromal cells (3). Counts for collagen-related genes such as Col1a1, Col1a2 and Col3a1, and Dcn (proteoglycan Decorin) were relatively high in PC2 (Supplementary Figure S3A), and the counts for genes coding for other extracellular matrix proteins, Fn1, Prg4 and Itga6, and Ccnd2 (cyclin CCND2) were high in PC1 (Supplementary Figure S3B and C). Although the gene expression profiles of PC3 and PC4 were grossly similar, that of Rn18s-rs5 (18S ribosomal protein-related sequence 5), which is important for protein synthesis (33), was markedly different (Supplementary Figure 3D, right). Also noteworthy is that expression of the Nestin protein-coding gene Nes was significantly high in PC1 (Supplementary Figure 3E, right), because Nestin+ MSCs form a niche for HSCs (22). In summary, the cells in the PC1 group expressed genes related to PC and HSC functions such as Vcam1, Nes and Il6, while those in both the PC1 and PC2 groups were grossly positive for gene expression of stem cell markers. On the other hand, the cells in PC3 and PC4 were characterized by roughly similar expression profiles of MSC markers as well as extracellular matrix components such as collagen, integrin and fibronectin. We concluded that our sort-purified PDGFRα+Sca-1+ MSCs supported BM PCs and were heterogenous in terms of the gene expression profiles. Discussion In this study, we investigated the in vitro effect of PDGFRα+Sca-1+ MSCs isolated from BM cells on the functional survival of BM PCs. We showed that the MSCs supported the survival of and antibody production by the PCs. We also showed that soluble factors including IL-6 played an important part in this support of PCs. While PC–BM stromal cell contact via a CD44 variant isoform was reported to induce IL-6 production by stromal cells (34), we failed to demonstrate a significant effect of the cell–cell contact on PC survival in this study. CD44 is involved in cell–cell and cell–extracellular matrix adhesion, and survival of IgG1-producing PCs was maintained on culture with anti-CD44 antibodies (10). CD44 is a hyaluronic acid receptor and interacts also with osteopontin, collagen and matrix metalloproteinases (MMPs) (35). CD44 itself is a surface marker of MSCs (14), and osteopontin and MMPs were detected in our culture supernatants on cytokine arraying. Although a whole spectrum of soluble factors and a potential cell–cell contact factor(s) in our PC–MSC culture system was not clarified, cell-adhesion molecules such as CD44 and related ligands could be involved in this system. Our scRNA-seq and PCA revealed that PDGFRα+Sca-1+ MSCs derived from BM comprise a heterogenous population, tentatively grouped into PC1–4 in terms of the gene expression profiles. Expression of Pdgfra and Ly6a was the highest in PC2 among them and it was also positive for other MSC markers. PC1 cells exhibited higher expression of Itgb1, Cd44 and Eng than PC2 ones did, whereas their expression of Itga5 and Thy1 was very low. Like PDGFRα+Sca-1− cells and PDGFRα−Sca-1+ cells, PC4 cells did not express stem cell markers, suggesting that they are going to undergo differentiation. PC3 cells seemed to have an intermittent gene expression profile between those of PC2 and PC4. On the basis of these observations, PC2 cells possessed most of the MSC profiles, while PC3 and PC4 cells were regarded as ones differentiating from MSCs. Then, the positioning of PC1 among these groups is obscure. The gene expression profiles of the extracellular matrix factors and others were different between PC1 and PC2. PC2 cells exhibited relatively high expression levels of collagen-related genes (Supplementary Figure S3A), while PC1 had high expression counts for Fn1, Prg4, Itga6 and Ccnd2 (Supplementary Figure S3B and C). These distinguishable profiles of PC1 and PC2 may be utilizable for further separation of these cell groups, which will enable us to clarify the positioning of PC1 cells. CXCL12+VCAM-1+ BM stromal cells, also known as CAR cells for CXCL12-abundant reticular cells, constitute a niche for BM PCs (3). It is widely accepted that the CXCL12–CXCR4 axis constituted primarily by CAR cells is important, but other CXCR4+ cells including eosinophils also cluster with the CXCL12+ stromal cells, thus forming a survival environment for PCs (4–9). These clustering cells secrete factors such as IL-6 and APRIL (10–12, 36). Because CAR cells are Sca-1− (37), our MSC preparation, which is Sca-1+, is different from CAR cells. Thus, our findings indicate that MSCs can also form a survival niche for BM PCs. On the other hand, our PCA study showed that VCAM-1+ cells and IL-6-producing cells are in PC1, but CXCL12-secreting cells in PC3. Although we do not have data regarding the spatial localization of these groups of MSCs in vivo, it is interesting to postulate that while CXCL12+ PC3 cells attract PCs in BM, IL-6-producing VCAM-1+ PC1 cells maintain their functional survival. Since IL-6 has also been shown to maintain the stemness of MSCs (38), PC1 cells may also contribute to maintenance of the stemness. Also, since Nestin+ MSCs were found in PC1 cells, it is suggested that HSCs and PCs are supported by PC1 cells together in BM. In this study, we showed a novel role of MSCs in BM for supporting the functional survival of PCs. Clarifying the molecular interactions between MSCs and BM PCs will provide us with an insight into the mechanism of long-term immunological memory as well as potential tools for regulating the memory. Funding Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15KK0328 and 17K15714 to A.I.-N. and 16H05201 and 17K19539 to T.T.), GSK Japan Research Grant 2015 and Naito Foundation Grant-in-Aid for Challenging Exploratory Research to A.I.-N., and Sumitomo Foundation to A.I.-N. and A.K. Conflicts of interest statement The authors declare no conflicts of interest. Acknowledgements We thank Mami Kikuchi, Makiko Nakagawa and Kiyotaka Kuroda (Tohoku University Graduate School of Medicine) for their technical assistance, and Nicholas Halewood for the editorial assistance. We also acknowledge the technical support of the Biomedical Research Core of Tohoku University Graduate School of Medicine. References 1 Ahmed , R. and Gray , D . 1996 . Immunological memory and protective immunity: understanding their relation . Science 272 : 54 . Google Scholar CrossRef Search ADS PubMed 2 Andraud , M. , Lejeune , O. , Musoro , J. Z. , Ogunjimi , B. , Beutels , P. and Hens , N . 2012 . Living on three time scales: the dynamics of plasma cell and antibody populations illustrated for hepatitis a virus . PLoS Comput. Biol . 8 : e1002418 . Google Scholar CrossRef Search ADS PubMed 3 Tokoyoda , K. , Egawa , T. , Sugiyama , T. , Choi , B. I. and Nagasawa , T . 2004 . Cellular niches controlling B lymphocyte behavior within bone marrow during development . Immunity 20 : 707 . Google Scholar CrossRef Search ADS PubMed 4 Chu , V. T. , Fröhlich , A. , Steinhauser , G. et al. 2011 . Eosinophils are required for the maintenance of plasma cells in the bone marrow . Nat. Immunol . 12 : 151 . Google Scholar CrossRef Search ADS PubMed 5 Rodriguez Gomez , M. , Talke , Y. , Goebel , N. , Hermann , F. , Reich , B. and Mack , M . 2010 . Basophils support the survival of plasma cells in mice . J. Immunol . 185 : 7180 . Google Scholar CrossRef Search ADS PubMed 6 Jinquan , T. , Jacobi , H. H. , Jing , C. et al. 2000 . Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4 . J. Allergy Clin. Immunol . 106 : 313 . Google Scholar CrossRef Search ADS PubMed 7 Winter , O. , Moser , K. , Mohr , E. et al. 2010 . Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow . Blood 116 : 1867 . Google Scholar CrossRef Search ADS PubMed 8 Rozanski , C. H. , Arens , R. , Carlson , L. M. et al. 2011 . Sustained antibody responses depend on CD28 function in bone marrow-resident plasma cells . J. Exp. Med . 208 : 1435 . Google Scholar CrossRef Search ADS PubMed 9 Glatman Zaretsky , A. , Konradt , C. , Dépis , F. et al. 2017 . T regulatory cells support plasma cell populations in the bone marrow . Cell Rep . 18 : 1906 . Google Scholar CrossRef Search ADS PubMed 10 Cassese , G. , Arce , S. , Hauser , A. E. et al. 2003 . Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals . J. Immunol . 171 : 1684 . Google Scholar CrossRef Search ADS PubMed 11 Benson , M. J. , Dillon , S. R. , Castigli , E. et al. 2008 . Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL . J. Immunol . 180 : 3655 . Google Scholar CrossRef Search ADS PubMed 12 Jourdan , M. , Cren , M. , Robert , N. et al. 2014 . IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors . Leukemia 28 : 1647 . Google Scholar CrossRef Search ADS PubMed 13 Minges Wols , H. A. , Underhill , G. H. , Kansas , G. S. and Witte , P. L . 2002 . The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity . J. Immunol . 169 : 4213 . Google Scholar CrossRef Search ADS PubMed 14 Houlihan , D. D. , Mabuchi , Y. , Morikawa , S. et al. 2012 . Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α . Nat. Protoc . 7 : 2103 . Google Scholar CrossRef Search ADS PubMed 15 Pittenger , M. F. , Mackay , A. M. , Beck , S. C. et al. 1999 . Multilineage potential of adult human mesenchymal stem cells . Science 284 : 143 . Google Scholar CrossRef Search ADS PubMed 16 Krampera , M. , Glennie , S. , Dyson , J. et al. 2003 . Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide . Blood 101 : 3722 . Google Scholar CrossRef Search ADS PubMed 17 Ren , G. , Zhang , L. , Zhao , X. et al. 2008 . Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide . Cell Stem Cell 2 : 141 . Google Scholar CrossRef Search ADS PubMed 18 Asari , S. , Itakura , S. , Ferreri , K. et al. 2009 . Mesenchymal stem cells suppress B-cell terminal differentiation . Exp. Hematol . 37 : 604 . Google Scholar CrossRef Search ADS PubMed 19 Schena , F. , Gambini , C. , Gregorio , A. et al. 2010 . Interferon-γ-dependent inhibition of B cell activation by bone marrow-derived mesenchymal stem cells in a murine model of systemic lupus erythematosus . Arthritis Rheum . 62 : 2776 . Google Scholar CrossRef Search ADS PubMed 20 Che , N. , Li , X. , Zhang , L. et al. 2014 . Impaired B cell inhibition by lupus bone marrow mesenchymal stem cells is caused by reduced CCL2 expression . J. Immunol . 193 : 5306 . Google Scholar CrossRef Search ADS PubMed 21 Luz-Crawford , P. , Djouad , F. , Toupet , K. et al. 2016 . Mesenchymal stem cell-derived interleukin 1 receptor antagonist promotes macrophage polarization and inhibits B cell differentiation . Stem Cells 34 : 483 . Google Scholar CrossRef Search ADS PubMed 22 Méndez-Ferrer , S. , Michurina , T. V. , Ferraro , F. et al. 2010 . Mesenchymal and haematopoietic stem cells form a unique bone marrow niche . Nature 466 : 829 . Google Scholar CrossRef Search ADS PubMed 23 El Haddad , N. , Heathcote , D. , Moore , R. et al. 2011 . Mesenchymal stem cells express serine protease inhibitor to evade the host immune response . Blood 117 : 1176 . Google Scholar CrossRef Search ADS PubMed 24 da Silva Meirelles , L. , Chagastelles , P. C. and Nardi , N. B . 2006 . Mesenchymal stem cells reside in virtually all post-natal organs and tissues . J. Cell Sci . 119 ( Pt 11 ): 2204 . Google Scholar CrossRef Search ADS PubMed 25 Phinney , D. G. , Kopen , G. , Isaacson , R. L. and Prockop , D. J . 1999 . Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation . J. Cell. Biochem . 72 : 570 . Google Scholar CrossRef Search ADS PubMed 26 Peister , A. , Mellad , J. A. , Larson , B. L. , Hall , B. M. , Gibson , L. F. and Prockop , D. J . 2004 . Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential . Blood 103 : 1662 . Google Scholar CrossRef Search ADS PubMed 27 Ohinata , Y. , Payer , B. , O’Carroll , D. et al. 2005 . Blimp1 is a critical determinant of the germ cell lineage in mice . Nature 436 : 207 . Google Scholar CrossRef Search ADS PubMed 28 Luckashenak , N. A. , Ryszkiewicz , R. L. , Ramsey , K. D. and Clements , J. L . 2006 . The Src homology 2 domain-containing leukocyte protein of 76-kDa adaptor links integrin ligation with p44/42 MAPK phosphorylation and podosome distribution in murine dendritic cells . J. Immunol . 177 : 5177 . Google Scholar CrossRef Search ADS PubMed 29 Hashimshony , T. , Senderovich , N. , Avital , G. et al. 2016 . CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq . Genome Biol . 17 : 77 . Google Scholar CrossRef Search ADS PubMed 30 Anderson , P. , Carrillo-Gálvez , A. B. , García-Pérez , A. , Cobo , M. and Martín , F . 2013 . CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities . PLoS One 8 : e76979 . Google Scholar CrossRef Search ADS PubMed 31 Stephan , R. P. , Reilly , C. R. and Witte , P. L . 1998 . Impaired ability of bone marrow stromal cells to support B-lymphopoiesis with age . Blood 91 : 75 . Google Scholar PubMed 32 Underhill , G. H. , Kolli , K. P. and Kansas , G. S . 2003 . Complexity within the plasma cell compartment of mice deficient in both E- and P-selectin: implications for plasma cell differentiation . Blood 102 : 4076 . Google Scholar CrossRef Search ADS PubMed 33 Rowe , L. B. , Janaswami , P. M. , Barter , M. E. and Birkenmeier , E. H . 1996 . Genetic mapping of 18S ribosomal RNA-related loci to mouse chromosomes 5, 6, 9, 12, 17, 18, 19, and X . Mamm. Genome 7 : 886 . Google Scholar CrossRef Search ADS PubMed 34 Van Driel , M. , Günthert , U. , van Kessel , A. C. et al. 2002 . CD44 variant isoforms are involved in plasma cell adhesion to bone marrow stromal cells . Leukemia 16 : 135 . Google Scholar CrossRef Search ADS PubMed 35 Ponta , H. , Sherman , L. and Herrlich , P. A . 2003 . CD44: from adhesion molecules to signalling regulators . Nat. Rev. Mol. Cell Biol . 4 : 33 . Google Scholar CrossRef Search ADS PubMed 36 Kometani , K. and Kurosaki , T . 2015 . Differentiation and maintenance of long-lived plasma cells . Curr. Opin. Immunol . 33 : 64 . Google Scholar CrossRef Search ADS PubMed 37 Sugiyama , T. and Nagasawa , T . 2012 . Bone marrow niches for hematopoietic stem cells and immune cells . Inflamm. Allergy Drug Targets 11 : 201 . Google Scholar CrossRef Search ADS PubMed 38 Pricola , K. L. , Kuhn , N. Z. , Haleem-Smith , H. , Song , Y. and Tuan , R. S . 2009 . Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism . J. Cell. Biochem . 108 : 577 . Google Scholar CrossRef Search ADS PubMed © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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International ImmunologyOxford University Press

Published: Feb 24, 2018

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