Human “Orchestrator” CD11b+ B1 Cells Spontaneously Secrete Interleukin-10 and Regulate T-Cell Activity
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Immune regulation produced by B cells has been attributed to production and secretion of interleukin (IL)-10, which is a characteristic of mouse B1 cells. In view of the widespread clinical use of B-cell depletion therapies in autoimmune and malignant diseases, it is important to monitor the function and fate of regulatory B cells. However, there is no consensus regarding the phenotypic identity of human IL-10+ B cells. Here we show that human CD11b+ B1 cells, one of two recently described subpopulations of B1 cells, spontaneously produce IL-10 and suppress T-cell activation. In view of the capacity of these B cells to either stimulate T-cell proliferation or suppress T-cell activation, CD11b+ B1 cells are considered to be capable of orchestrating elements of immune responsiveness and thus are termed “orchestrator B1 cells,” or “B1orc,” whereas CD11b− B1 cells that primarily secrete antibody are termed “secretor B1 cells,” or “B1sec.”
We identified the phenotype of human B1 cells as CD20+CD27+CD43+ (1). Human B1 cells are found both in umbilical cord blood and adult peripheral blood. Human B1 cells share with mouse B1 cells specificity for certain microbial and self-antigens. We recently described phenotypic and functional subdivision of human B1 cells: CD11b− B1 cells secrete substantial amounts of antibody but stimulate T cells less efficiently than CD11b+ B1 cells; CD11b+ B1 cells express elevated levels of CD86 and stimulate T cells strongly but secrete less antibody than CD11b− B1 cells (2). Most human B1 cells are CD11b−, whereas a smaller proportion are CD11b+, although this latter population is markedly increased in patients with lupus disease.
The ability of the immune system to reign in the inflammatory response and limit it to an appropriate level is critical to addressing potential immunemediated damage. Failure to adequately regulate immune system activity is implicated in multiple clinical situations, including inflammatory bowel disease, respiratory asthma, food allergies, autoimmune diseases and transplant rejection. Elucidation of the cells responsible for immune regulation is expected to be invaluable in advancing the understanding of these and other diseases.
Recent work has led to the recognition that certain B cells can be regulatory in nature (Breg) and can suppress inflammatory and autoreactive responses (3). It is conceivable, then, that the current broad B-cell depletion therapies being used therapeutically could erode an important B cell-derived modulatory influence. Thus, it is of great importance to understand how to recognize Breg cells so that their number can be monitored and their fate can be determined. Immune regulation by B cells has been attributed to production and secretion of IL-10, and it has long been known that IL-10 is produced by murine B-1 cells (4,5). More recently, IL-10+ mouse B cells were characterized as CD1dhi in addition to CD5+ and may include B-1 cells, MZ B cells and T2-MZ precursors (6). Comparable studies with human B cells have yielded diverse results. Human B cells that produce IL-10 have been reported to be enriched within the CD24hiCD38hi gated population, or the CD24hiCD27+ gated population, but also have been shown to be present among both CD24low and CD38low/− populations (5,7,8). It is notable that these reports involve the use of stimulatory regimens before assessment of IL-10 content, which could induce secretion in B cells that were not previously secreting spontaneously (5).
Moreover, the majority of the putative B-cell populations possessing B regulatory activity via secretion of IL-10, both CD24hiCD38hi cells and CD24hiCD27+ cells, are negative for production of IL-10, as shown by intracellular staining. Only 10–15% of CD24hiCD38hi cells are producing IL-10 after several days of exposure to a stimulatory regimen (8), whereas only 3–4% of CD24hiCD27+ cells are producing IL-10 after brief stimulation (7). Thus, the level of regulatory B-cell enrichment that can be obtained with these markers is severely limited. An additional issue is that neither of these gating strategies identifies a unique phenotype for IL-10-secreting cells, since the CD24hiCD38hi gate includes transitional cells as well as naive B cells, whereas the CD24hiCD27+ gate includes true memory cells, circulating marginal zone (MZ) cells and B1 cells.
Because CD11b+ B1 cells are capable of driving T-cell proliferation through surface expression of CD86, we questioned whether members of this subpopulation might also be capable of modulating immune cell activity via production and secretion of IL-10, like mouse B1 cells. We found that human CD11b+ B1 cells do spontaneously produce and secrete IL-10 that is capable of downregulating T-cell responses.
Materials and Methods
Donors and Samples
Adult peripheral blood samples were obtained by venipuncture of adult volunteers after obtaining informed consent in accordance with the Declaration of Helsinki. Additional samples in the form of leukopacks were obtained from the New York Blood Center on the day of donation. This study was approved by, and all samples were obtained in accordance with, the Institutional Review Board of the North Shore-LIJ Health System.
All blood samples were treated in a similar manner and processed promptly on receipt. Mononuclear cells were obtained by density gradient separation using lymphocyte separation medium (Cellgro, Manassas, VA, USA). Except as otherwise noted, mononuclear cells were then washed and resuspended in RPMI 1640 (Cellgro) containing 10% fetal calf serum plus 2 mmol/L l-glutamine, 10 mmol/L HEPES (pH 7.25), 100 U/mL penicillin and 100 µg/mL streptomycin.
For some experiments, B cells were enriched by CD19+ selection using the EasySep Human CD19+ B Cell magnetic bead selection kit (STEMCELL Technologies Inc., Vancouver, BC, Canada) according to the manufacturer’s instructions.
Flow Cytometry Analysis and Cell Sorting
Enriched B cells and mononuclear cells were sort-purified on an Influx instrument (BD Biosciences, San Jose, CA, USA) after immunofluorescent staining, as described in Results. In all experiments displayed, CD20+ cells were studied. Flow cytometric analysis of immunofluorescently stained cells was carried out on an LSR-II instrument (BD Biosciences). When intracellular staining was performed, cells were fixed in 1.6% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with 0.2% Tween for 15 min at 37°C.
Enzyme-Linked Immunosorbent Assay
B cells were cultured in RPMI medium for 5 d, after which supernatant fluid was assayed for IL-10 by enzyme-linked immunosorbent assay (ELISA; BD Biosciences) according to the manufacturer’s directions.
T-Cell Activation Assays
Sort-purified CD19-enriched B cells were cocultured with negatively selected CD4+ T cells (STEMCELL Technologies Inc.), 1:1 in 100 µL RPMI medium (1,2) in the presence of plate-bound anti-CD3 monoclonal antibody (0.5 µg/mL) for 72 h. T-cell expression of tumor necrosis factor (TNF)-α was assessed by intracellular staining after treatment with phorbol myristate acetate (PMA) and ionomycin plus Golgi-stop for the last 6 h. IL-10 neutralization experiments were performed by preincubating B cells with IL-10 neutralizing antibody for 30 min before introduction of B cells into the T-cell coculture.
Anti-CD20-V450, anti-CD27-APC, anti-CD43-FITC, anti-CD11b-PE and anti-CD11b-PerCP, anti-CD3-PE, anti-CD19-PE-Cy7, anti-CD4-PerCP-Cy5.5, anti-TNF-α-APC and anti-CD24-PE were obtained from BD Biosciences, along with MsIgG1κ isotype controls conjugated to V450, APC, FITC, PE and PerCP-Cy5.5, plus MsIgG2aκ isotype controls conjugated to PE and PerCP. Anti-IL-10 eFluor 450 was obtained from eBiosciences. IL-10 neutralizing antibody was obtained from R&D Systems (Minneapolis, MN, USA).
Statistical analysis was performed using Prism Software, version 5.0d, for Mac (Graphpad Software Inc., La Jolla, CA, USA). Data are displayed when appropriate as mean plus or minus the standard error of the mean (SEM). Data were compared for statistically relevant differences by using Student’s t test with two-tailed analysis. P values are shown in relevant figures.
Tedder and colleagues examined IL-10+ B cells after brief stimulation with PMA and ionomycin plus brefeldin (7). They reported that such B cells are defined by CD24hiCD27+. With these criteria, they obtained an approximately fourfold enrichment of IL-10+ B cells from 0.8% of all B cells to 3.4% of CD24hiCD27+ B cells. We plotted CD3−CD20+ B cells according to their gating scheme and generated a similar division of adult peripheral blood B cells into CD24hiCD27+ and C24loCD27− populations; however, we also identified smaller numbers of CD24− B cells (Figure 6C). When we displayed CD11b+ B1 cells on the same plot, we found a little more than half phenotyped as CD24hiCD27+; however, the level of enrichment is limited by the large number of true memory and other B cells contained within that gate, amounting to about one-third of all B cells (Figure 6D). Thus, the CD24hiCD27+ gating parameters offered by Iwata et al. (7), although inclusive of many CD11b+ B1 cells, still omit a substantial fraction and at the same time are not particularly specific for CD11b+ B1 cells. For these reasons, gating for CD24hiCD27+ B cells provides only modest enrichment of CD11b+ B1 cells and is not a practical guide to identifying or enriching these cells.
We have shown here that human CD11b+ B1 cells spontaneously produce and secrete IL-10 and suppress T-cell activation. Inasmuch as a high percentage of CD11b+ B1 cells stain positive for IL-10, and CD11b+ B1 cells represent a very small proportion of all B cells, focusing on CD11b+ B1 cells leads to very high enrichment for IL-10 positivity, which far exceeds that reported from a focus on CD24hiCD38hi or CD24hiCD27+ B cells, as suggested elsewhere (7,8). It is important to note that, in these latter studies, B cells were stimulated with PMA plus ionomycin for 5–6 h before assay, and in some cases, with additional regimens for longer periods of time. Inasmuch as B cells would not be exposed to PMA or ionomycin in vivo, examination of B cells thus stimulated may be difficult to translate into what is actually occurring in vivo. Our approach differs in that B cells were unstimulated and IL-10 expression was, as far as can be determined, truly spontaneous. This work furthers the understanding of how effector functions of the immune system can be regulated by B cells in general and by B1 cells in particular.
Our results indicate that IL-10 is associated with a distinct identified B-cell subset in the human system that expresses CD20, CD27, CD43 and CD11b (CD11b+ B1 cells). A majority of these cells produce IL-10, in contrast to the B cells identified in the CD24hiCD38hi or CD24hi CD27+ B-cell gating schemes reported by others, which miss many IL-10+ B cells and which include heterogeneous groupings of cells, of which only a small proportion express IL-10 (7,8). An interesting and important question is whether IL-10 production and B-cell regulatory function is limited to CD11b+ B1 cells. In our work, removal of CD11b+ B1 cells eliminated B cells that express IL-10 (Figure 2) as well as B cells that possess regulatory function (Figure 3). However, in the well-studied murine system, although B-1 cells are the main source of B cell-derived IL-10, they are not the sole source (4,6). It is notable that some key characteristics of murine B-1 cells are recapitulated by stimulated B-2 cells. In that sense, our results speak only to homeostatic production of IL-10 in the absence of exogenous manipulation or stimulation, but do not rule out the possibility that other B-cell populations secrete IL-10 and suppress immune responses after activation in vivo.
IL-10 is spontaneously produced by unstimulated CD11b+ B1 cells, and immunosuppression by CD11b+ B1 cells is dependent on IL-10. This does not mean that the former represents the sum and substance of the latter. We have observed that various stimulatory regimens can increase the amount of IL-10 secreted by CD11b+ B1 cells and thus cannot dismiss the idea that individual CD11b+ B1 cells may increase their secretion of IL-10 above the level they produce spontaneously and that this may increase regulatory function. Conversely, although IL-10 may be the dominant means of B-cell regulatory function, not all B-cell regulatory function is mediated through secretion of IL-10 (10, 11, 12, 13).
A number of clinical conditions are linked to dysregulation of IL-10. Patients suffering from asthma have lower levels of serum IL-10 as well as lower production of IL-10 from immune cells (14). Further, lower levels of IL-10 and IL-10 gene polymorphisms appear to correlate with disease severity in asthma and α1-antitrypsin deficiency as well as other allergic conditions (15,16). Moreover, inflammatory conditions affecting the gut, such as Crohn’s disease and inflammatory bowel disease, are associated with defects in immune regulation and IL-10 in particular (17,18). From the therapeutic standpoint, it is critically important to discern the identity and role of regulatory B cells in patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosis, psoriasis and Sjögren’s Syndrome who undergo treatment with B-cell depletion therapy, lest beneficial modulatory B cells be removed along with pathogenic B cells. Thus, understanding the origin of immunoregulatory IL-10 and determining the role of regulatory B cells are key issues for clinical medicine.
Previous work showed that human B1 cells are divided on the basis of surface CD11b expression and demonstrated that CD11b+ B1 cells drive T-cell proliferation due, at least in part, to elevated expression of the costimulatory molecule CD86. Here we show that this same CD11b+ B1 cell population secretes IL-10 and suppresses CD3-mediated T-cell activation. In view of the capacity of CD11b+ B1 cells to stimulate T-cell proliferation and to modulate T-cell activation, these B1 cells appear capable of orchestrating aspects of immune responsiveness. We propose that CD11b+ B1 cells that directly affect T-cell activation and proliferation be termed “orchestrator B1 cells,” or “B1orc,” and that CD11b− B1 cells, which spontaneously secrete antibody but have little effect on T cells, be termed “secretor B1 cells” or “B1sec.”
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
The authors gratefully acknowledge support from the National Institutes of Health (grant AI29690) and the Zucker Family Foundation.
DO Griffin and TL Rothstein designed the study. DO Griffin performed the experiments. DO Griffin and TL Rothstein analyzed the data and contributed to the final manuscript.
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