Systematic evaluation of the conditions required for the generation of immature rat bone marrow-derived dendritic cells and their phenotypic and functional characterization

This study systematically evaluated the conditions required for generating immature rat bone marrow-derived dendritic cells (BMDCs) and characterized their phenotype. The culture of Wistar rat bone marrow cells for 7 days in an optimal cytokine environment (granulocyte macrophage-colony stimulating factor (GM-CSF), 10 ng/ml; IL-4, 5 ng/ml) resulted in adherent and non-adherent cell populations, but only the adherent population predominantly expressed the rat DC marker OX62. Adherent OX62+ cells were immature, in that they expressed lower levels of CD86 and MHC class II and were more phagocytic than their non-adherent OX62+ counterparts. Adherent BMDCs constitutively produced low levels of IL-12 and nitric oxide (NO), levels of both of which were markedly increased following lipopolysaccharide (LPS) activation. Activation also increased the proportion of OX62+ cells expressing CD40, CD54 and CD86 and their intensity of expression, however, unlike murine BMDCs, it had no effect on CD80 and MHC class II expression. Although the proliferation of allogeneic Lewis splenocytes in response to immature resting and LPS-activated (mature) Wistar BMDCs was of a similar magnitude, levels of IL-12 after 5 days were significantly higher in cultures containing LPS-activated BMDCs and the IFN-gamma/IL-4 cytokine ratio differed markedly (2.35 vs. 6.66, respectively). This study systematically defines conditions for generating immature rat BMDC populations and demonstrates qualitative differences in the phenotype of immune responses induced by resting and LPS-activated BMDC populations.     

IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity

Dendritic cells (DC) of myeloid origin can be generated from mouse bone marrow (BM) using granulocyte macrophage-colony stimulating factor (GM-CSF). Immature major histocompatibility complex (MHC) II(low) DC are known to bear a high endocytosis capacity, in contrast to DC precursors and mature DC. Now we found that a subset of MHC II(low) DC in BM-DC cultures is unable to exert mannose receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (DX) and resembles immature Langerhans cells (LC). The FITC-DX endocytosis activity of LC-like cells occurs at an earlier stage of development, where the surface MHC II expression is absent or very weak. This LC-like subset expresses higher levels of E-cadherin but lower amounts of the markers Gr-1, scavenger receptor 2F8, and CD11b, when compared with the highly endocytic DC subset. The latter myeloid DC resemble monocyte-derived DC (MoDC). The sorted LC-like population develops completely and exclusively into mature MHC IIhigh DC, and the MoDC-like cells remain immature MHC II(low) DC or develop into adherent MHC IIneg macrophages or mature into MHC IIhigh DC. The development of LC-like cells is promoted by interleukin-4. Thus, we show here that the simultaneous development of LC-like and MoDC-like DC subsets occurs in standard bulk cultures with GM-CSF, suggesting the existence of two different precursors for LC and MoDC in BM.     

Freezing and thawing of bone marrow-derived murine dendritic cells with subsequent retention of immunophenotype and of antigen processing and presentation characteristics

Murine dendritic cells (DCs) are widely used for experimental vaccinations in mouse models. A high-yield method for freezing and thawing batches of these cells, if compatible with retention of cell immunophenotype, would reduce the time required for repeated preparations from DC precursors in bone marrow (BM), as well as variability among lots. Following depletion of specific lineages, murine bone marrow cells from C57BL/6 inbred-strain mice were grown in medium containing 10% fetal calf serum (FCS) and granulocyte/macrophage colony-stimulating factor (GM-CSF); after 6 days, large numbers of immature DCs were obtained. The immature cells were frozen in complete medium with GM-CSF and 10% DMSO, at a cell density of 5x10(6) DCs/ml. After thawing, 80% of DCs survived; they were induced to mature by addition of lipopolysaccharide (LPS). In comparison with fresh DCs, the thawed DCs had similar morphology, purity, and expression of class I (H-2D(b) and H-2K(b)) and class II major histocompatibility complex (MHC) proteins, as well as CD11b, CD11c, CD40, CD80, and CD86 molecules. Freeze-thawing did not affect trafficking to T cell areas of spleen, nor reduce the capacity to stimulate an alloresponse. Frozen-thawed cells were also proficient at uptake, processing, and presentation of native or denatured ovalbumin (OVA) protein to a peptide-specific T cell hybridoma, and were able to induce T cell responses in vivo after being loaded with denatured OVA protein. The ability to freeze and thaw DCs, and to obtain high yields without altering their essential properties, will facilitate future immunotherapy experiments in laboratory mouse models.     

Myeloid dendritic cell precursors generated from bone marrow suppress T cell responses via cell contact and nitric oxide production in vitro

Tolerogenic activity of myeloid dendritic cells (DC) has so far been attributed mostly to immature or semi-mature differentiation stages but never to their precursor cells. Although myeloid suppressor cells (MSC) have been isolated ex vivo, their developmental relationship to DC and their precise phenotype remained elusive. Here, we describe the generation of MSC as myeloid DC precursors with potent suppressive activity on allogeneic and OVA-specific CD4+ and CD8+ T cell responses in vitro. These MSC appear transiently in DC cultures of bone marrow (BM) cells after 8-10 days under low GM-CSF conditions or after 3-4 days under high GM-CSF conditions. They represent CD11c- myeloid precursor cells with ring-shaped nuclei and are Gr-1low (i.e. Ly-6C+, Ly-6Glow), CD11b+, CD31+, ER-MP58+, asialoGM1+ and F4/80+. Sorted MSC develop into CD11c+ DC within 6 days. Their suppressor activity partially depends on IFN-gamma stimulation. Suppression is mediated through mechanisms requiring cell contact and nitric oxide but is independent of TNF, CD1d and TGF-beta. Together, our data describe the generation of MSC with distinct suppressor mechanisms in vitro preceding their development into immature DC.     

4种细胞因子组合方式对小鼠树突状细胞分化发育的影响

目的观察在体外培养时4种细胞因子(CK)组合方式对小鼠骨髓源树突状细胞(DC)分化、增殖、发育的影响.方法用不同的CK定向诱导小鼠骨髓细胞分化为DC,通过流式细胞仪(荧光抗体双标记法)测定CD11c+细胞比例、MHC-Ⅱ类分子的表达及在脂多糖(LPS)刺激后CD86表达的变化.结果GM-CSF+IL-3+SCF促进DC分化、增殖的能力明显高于其他3组(P＜0.05).该组CK所诱导的DC在LPS刺激后,CD86表达增加的幅度明显低于GM-CSF+IL-4组(P＜0.01).结论GM-CSF、IL-3和SCF对于促进小鼠骨髓细胞向DC定向分化、增殖有协同作用,分化后的DC多数处于发育早期,DC前体所占的比例较大.     

Maturation of antigen-presenting cells is compromised in HLA-G transgenic mice

The human MHC class Ib antigen HLA-G is thought to regulate maternal immune responses during pregnancy. Here we show that expression of HLA-G in transgenic mice diminished cellular immunity by inhibiting maturation of myelomonocytic cells into functional antigen-presenting cells (APC). Skin allografts applied to HLA-G transgenic mice survived longer and resultant T cell responses were less potent compared to control mice. T cells from HLA-G mice responded normally to allogeneic APC and immunohistological analyses of spleen revealed no marked abnormalities. However, spontaneous outgrowths of myeloid cells were observed when bone marrow or splenocytes from HLA-G mice were cultured in vitro, but functionally competent APC did not develop spontaneously in bone marrow cultures supplemented with granulocyte macrophage colony stimulating factor (GM-CSF). Addition of lipopolysaccharide (LPS) to GM-CSF-derived bone marrow cultures rescued APC maturation. Studies using HLA-G tetrameric reagents revealed that HLA-G-specific binding activity was associated with CD11c(+) myelomonocytic cells, while binding to lymphoid and NK cell subsets was undetectable. These data show that spontaneous maturation of functionally competent dendritic cells (DC) is compromised in HLA-G mice. We hypothesize that HLA-G inhibits maturation of DC via receptor-mediated interactions with myelomonocytic precursors, which render immature DC precursors unable to receive signals from activated T cells.     

Expression of milk fat globule epidermal growth factor 8 in immature dendritic cells for engulfment of apoptotic cells

Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein that stimulates the engulfment of apoptotic cells by phagocytes. Here, we show that mouse immature dendritic cells (DC) generated in vitro by culturing bone marrow progenitors in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), and Langerhans cells present in the skins, expressed MFG-E8. Bone marrow-derived macrophages generated by M-CSF did not express MFG-E8. MFG-E8 expressed in immature DC was found to be secreted as exosomes. The expression of MFG-E8 was significantly suppressed when the immature DC were induced to mature by treating them with lipopolysaccharides. This expression of MFG-E8 was well correlated with the ability of the cells to engulf apoptotic cells. That is,immature DC phagocytosed apoptotic cells more efficiently than did mature DC or bone marrow-derived macrophages. The ability of immature DC to engulf apoptotic cells was severely reduced when the immature DC were prepared from MFG-E8-deficient mice. These results indicated that MFG-E8 plays an essential role in the engulfment of apoptotic cells by bone marrow-derived immature DC.     

A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells

We established a novel culture method for generating dendritic cells (DC) from mouse bone marrow (BM) cells. Unfractionated bulk BM cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5-7 days and a DC population was isolated by gradient centrifugation with 14.5% (w/v) metrizamide. Through this method, 30-40 x 10(6)/mouse DC with 85-95% purity was obtained on day 7; this yield was higher than those of conventional DC generated by Inaba's method either with GM-CSF alone (conventional-GM DC) or GM-CSF and IL-4 (conventional-GM/4 DC). Bulk-cultured DC have a more matured phenotype than both conventional-GM and -GM/4 DC as shown by higher expression of CD86, MHC class II and CD40. Functional analyses reveal that (1) bulk-DC show less ability in endocytosis than conventional-GM DC and are comparable in IL-12 p70 production with conventional-GM and -GM/4 DC. (2) Bulk-DC exhibit stronger stimulatory capacity in allogeneic T-cell proliferation than conventional DC. (3) By using ovalbumin (OVA) and OVA-specific T-cell receptor (TCR) transgenic mice (DO11.10) system, OVA protein-loaded bulk-DC stimulated CD4 T cells of DO11.10 mice more than conventional-GM DC and comparable with conventional-GM/4 DC. (4) Furthermore, OVA peptide-pulsed bulk-DC stimulated CD4 T cells more than conventional-GM and -GM/4 DC. These data indicate that bulk-DC are functionally more mature than conventional DC. Taken together, bulk-culture method is a simple technique for generating functionally mature BM-DC in large quantities and high purity.     

IL-17A 协同GM-CSF 和LPS 促进骨髓细胞衍生树突状细胞的分化和成熟研究

目的:探讨IL鄄17A 对小鼠骨髓细胞衍生树突状细胞分化和成熟的影响。方法:分离小鼠骨髓细胞,加入含GM-CSF(20 ng/ ml)RPMI1640 完全培基培养8 d,诱导小鼠骨髓单个核细胞向DC 分化,加入LPS(1 滋g/ ml)继续培养36 h,进一步诱导DC 成熟,同时在骨髓细胞衍生诱导DC 分化及成熟的不同阶段加入不同浓度的rmIL-17A(10、100 ng/ ml),采用流式细胞术检测DC 表面共刺激分子的表达,ELISA 方法检测DC 培养上清中IL-12p40 和IL-10 水平。结果:rmIL-17A 可促进GM-CSF 诱导骨髓细胞衍生DC 表面共刺激分子CD40、CD80、CD86 和MHC域的表达,且具有剂量依赖性,其中以高浓度rmIL-17A刺激组的CD40 及MHC域表达增加最显著;在LPS 诱导DC 成熟阶段加入rmIL-17A,骨髓细胞衍生DC 共刺激分子CD40、CD80、CD86 和MHC域的表达均明显增加,并且随着rmIL-17A 浓度的增加,CD86 和MHC域的表达水平也随之增高;同时与未加rmIL鄄17A 的对照组相比,低浓度rmIL-17A 组LPS 刺激骨髓细胞衍生DC 分泌IL-12p40 和IL鄄10 水平均显著增加(P <0.001),高浓度rmIL-17A 组IL-12p40 水平显著增高(P<0.001),但IL-10 水平没有变化。结论:IL-17A 可促进GM-CSF 诱导的骨髓细胞衍生DC 前体细胞表型发展,并能协同LPS 诱导骨髓衍生DC 的分化和成熟。     

Granulocyte precursors are the principal cells in bone marrow that stimulate allospecific cytolytic T-lymphocyte responses.

Mouse bone marrow cells were fractionated and enriched for functional activity as stimulators of allospecific cytolytic T-lymphocyte (CTL) responses in vitro. The relevant stimulator cells were enriched sequentially in the low-density fraction of bone marrow, its 2-hr adherent and 18-hr non-adherent fractions and in the FcR-negative fraction of 18-hr non-adherent cells. The functionally enriched cell population contained over 90% granulocyte precursors by ultrastructural analysis. The results indicate that granulocyte precursors are the principal cells in bone marrow that stimulate alloreactive T-cell responses.     

Generation or large numbers of immature and mature dendritic cells from rat bone marrow cultures

We have defined conditions for generating large numbers of dendritic cells (DC) in marrow cultures from 10 – 12-week-old ACI or WF rats. The combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α, known to induce DC from human CD34⁺ progenitors, was not effective with rat. In contrast, GM-CSF plus IL-4 generated DC in high yield, corresponding to 30 – 40 % of the initial number of plated marrow cells. The DC proliferated in distinctive aggregates, in which most cells had an immature phenotype marked by undetectable surface B7 and high levels of MHC class II products within intracellular lysosomes. When dislodged and dispersed, the aggregates gave rise to mature stellate DC with abundant surface MHC class II and B7, sparse MHC class II^– lysosomes, and strong T cell-stimulating capacity. Therefore, rat marrow progenitors can generate large numbers of immature DC, with abundant intracellular MHC class II compartments, and potent, stimulatory, mature DC.     

Matched skin and sentinel lymph node samples of melanoma patients reveal exclusive migration of mature dendritic cells

Mature and immature myeloid dendritic cells (DCs) are thought to differentially modulate T-cell responses in secondary lymphoid tissues. Although mature DCs are believed to induce T-cell activation under proinflammatory conditions, immature DCs are believed to maintain a state of T-cell tolerance under steady state conditions. However, little is known about the actual activation state of human DCs under these different conditions. Here, we compare the frequency and activation state of human DCs between matched skin and sentinel lymph node (SLN) samples, after intradermal administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or saline, at the excision site of stage I primary melanoma. Although DCs remained immature (CD1a+CD83-) and mostly situated in the epidermis of the saline-injected skin (fully consistent with a quiescent steady state), mature (CD1a+CD83+) DC frequencies significantly increased in the GM-CSF-injected skin and correlated with the number of mature DCs in the SLN, indicative of increased DC migration. Interestingly, irrespective of GM-CSF or saline administration, all CD1a+ myeloid DCs in the SLN were phenotypically mature (ie, CD83+). These data are indicative of migration of small numbers of phenotypically mature DCs to lymph nodes under steady state conditions.     

Dendritic cell development in long-term spleen stromal cultures

The cellular microenvironments in which dendritic cells (DCs) develop are not known. DCs are commonly expanded from CD34+ bone marrow precursors or blood monocytes using a cocktail of growth factors including GM-CSF. However, cytokine-supported cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs that undergo limited proliferation and survive for only a short period of time. This lab has developed a long-term culture (LTC) system from spleen which readily generates a high yield of DCs. Hematopoietic cells develop under more normal physiological conditions than in cultures supplemented with cytokines. A spleen stromal cell monolayer supports stem cell maintenance, renewal, and the specific differentiation of only DCs and no other hematopoietic cells. Cultures maintain continuous production of a small population of small-sized progenitors and a large population of fully developed DCs. Cell-cell interaction between stromal cells and progenitor cells is critical for DC differentiation. The progenitors maintained in LTC appear to be quite distinct from bone marrow-derived DC progenitors that respond to GM-CSF. The majority of cells produced in LTC are large-sized cells with a phenotype reflecting myeloid-like DC precursors or immature DCs. These cells are highly endocytotic and weakly immunostimulatory for T cells. This model system predicts in situ production of DCs in spleen from endogenous progenitors, as well as a central role for spleen in DC hematopoiesis.     

CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock-in mouse.

The homing properties of subsets of lymphocytes and dendritic cells (DC) are regulated in part by the profile of chemokine receptors expressed. To determine how CCR6 influences cell trafficking, a mutant allele of the mouse CCR6 gene was produced that includes an enhanced green fluorescent protein (EGFP) reporter under the control of the CCR6 promoter. In mice heterozygous for the EGFP/CCR6 knock-in, CCR6 expression was detected on all mature B cells, subpopulations of splenic CD4(+) and CD8(+) T cells, and on some CD11c(+) DC. Most CD11b(+) myeloid DC expressed CCR6, but CD8alpha(+) lymphoid DC were negative for CCR6. Among myeloid DC, the CD4(+) subset was uniformly positive for CCR6 expression and the CD4(-) subset was mostly CCR6 positive. Epidermal Langerhans cells (LC) also expressed CCR6, but at lower levels than splenic myeloid DC. Culture of bone marrow precursors from the knock-in mice with GM-CSF for 4 to 6 days led to the appearance of a subset of CD11c(+) DC expressing CCR6. The differences in CCR6 expression among the major DC subsets indicate that CCR6 and its chemokine ligand MIP-3alpha participate in determining the positioning of DC subsets in epithelial and lymphoid tissues.     

Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression

Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c+ MHC II(NEG), CD11c+ MHC II(INT), CD11c+ MHC II(HI) expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O2 tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O2 or conventional conditions, and MDCs from low O2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O2 and conventional culture conditions were similarly potent allostimulatory APCs. The O2 tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.     

Limitations with in vitro production of dendritic cells using cytokines

Dendritic cells (DC) are the most effective antigen-presenting cells. Many studies now show that DC can be generated in vitro from a number of starting cell populations containing hematopoietic precursors. The protocols used involve different combinations of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF), which supports myeloid precursors, or interleukin-7, which supports lymphoid precursors. DC are commonly generated by in vitro culture of bone marrow or monocytes with GM-CSF and other cytokines. However, these cultures do not sustain DC production for long periods of time and do not allow the identification or study of intermediate stages in cell development. In vitro cytokine-dependent cultures of DC precursors do provide a reliable source of DC for stimulating immune responses. However, use of cells produced in cytokine-dependent cultures for the study of DC differentiation is limited, as DC development in vivo differs in cytokine dependency.     

CD38 expression in early B-cell precursors contributes to extracellular signal-regulated kinase-mediated apoptosis

CD38 is a 45 000 molecular weight transmembrane protein that is expressed in immature and mature lymphocytes. However, the expression and function of CD38 during B-cell differentiation in mice is poorly understood. Here, we report that CD38 is expressed from the earliest stages of B-cell development. Pre-pro-B, pro-B, pre-B and immature B cells from murine bone marrow all stained positive for CD38. Interestingly, CD38 expression increases with B-cell maturation. To assess the role of CD38 during B-cell maturation, CD38-deficient mice were analysed. CD38^−/− mice showed a significant increase in both the frequency of B-lineage cells and the absolute numbers of pre-pro-B cells in bone marrow; however, no other differences were observed at later stages. CD38 cross-linking in Ba/F3 cells promoted apoptosis and marked extracellular signal-regulated kinase (ERK) phosphorylation, and these effects were reduced by treatment with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and similar effects were observed in B-cell precursors from bone marrow. These data demonstrate that B-cell precursors in mouse bone marrow express functional CD38 and implicate the early ligation of CD38 in the ERK-associated regulation of the B-lineage differentiation pathway.     

Interferon γ Stimulates Cellular Maturation of Dendritic Cell Line DC2.4 Leading to Induction of Efficient Cytotoxic T Cell Responses and Antitumor Immunity

Dendritic cells （DCs） are the most potent antigen-presenting cells （APCs） for the initiation of antigen （Ag）-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-α for 7-10 days and stimulated with lipopolysaccharide （LPS）. However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class Ⅱ and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli （IL-1β, IFN-γ, TNF-α CpG and LPS） in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-γ was able to stimulate the maturation of DC2.4 cells. The IFN-γ-activated ovalbumin （OVA）-pulsed DC2.4 cells have capacity to upregulate MHC class Ⅱ, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8^＋ T cell responses and antitumor immunity. Therefore, IFN-γ-activated immortal DC2.4 ceils may prove to be useful in the study of DC biology and antitumor immunity.     

Development of dendritic cells from GM-CSF-/- mice in vitro : GM-CSF enhances production and survival of cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/- and wild type mice were established and compared for cell production. GM-CSF-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

A phenotypically distinct subset of immature B cells exhibits partial activation, increased survival, and preferential expression of VhS107

We have observed that immature B cells (IgM(low)IgD(-)) in the bone marrow of adult BALB/c mice exhibit heterogeneity, with a distinct subpopulation ( approximately 4-10%) expressing the CD43/S7 surface protein. These CD43/S7(+) immature B cells often express other surface antigens associated with B cell activation (CD5, CD11b, PD-1). Generation of optimal numbers of CD43/S7(+) immature B cells requires expression of a functional Btk protein, consistent with activation as a requisite for the CD43/S7(+) immature B cell phenotype. Like typical CD43/S7(-) immature B cells, the CD43/S7(+) immature B cells are predominantly resting cells, which are derived from cycling bone marrow B cell precursors. The CD43/S7(+) immature B cell population exhibits enhanced survival in vivo upon administration of the apoptosis-inducing corticosteroid, dexamethasone. Finally, CD43/S7(+) immature B cells show a fourfold increase in incidence of VhS107 micro heavy chain expression compared to the CD43/S7(-) immature B cells. Therefore, in adult murine bone marrow, the presence of a phenotypically distinct immature B cell population can be demonstrated which has undergone partial activation leading to increased survival and BCR-dependent Vh repertoire selection.