Human in vivo-induced spontaneous IgG-secreting cells from tonsil, blood and bone marrow exhibit different phenotype and functional level of maturation.

The subset of human spontaneous IgG-secretion cells consists of mature B lymphocytes which are capable of active and high rate IgG production in vitro without the need for additional stimuli. Therefore, such a cell subset provides a useful model for studying the terminal stages of B-cell maturation. The present work analyses the phenotypic and functional characteristics of spontaneous IgG secreting cells obtained from tonsil, blood and bone marrow. The tonsilar cell subset was CD9+ CD20+ CD19+ CD38+/-, the blood cell subset CD9- CD20- CD19+ CD38+/- and bone marrow cells were CD9- CD20- CD19+/- CD38+. The three cell subsets required de novo RNA and protein synthesis for IgG secretion to occur. Tonsilar and blood, but not bone marrow, subsets also required DNA synthesis to undergo IgG secretion. Kinetics studies revealed that IgG production by tonsil and blood cells reached a plateau after 3 days of culture. In contrast, the bone marrow cell subset secreted IgG in a linear fashion for 2 weeks. These results indicate that spontaneous IgG-secreting cells from different organs exhibit functional and phenotypic heterogeneity.     

Increase of CD57+ T cells in knee joints and adjacent bone marrow of rheumatoid arthritis (RA) patients: implication for an anti-inflammatory role

The distribution of CD57⁺ T and CD56⁺ T cells in patients with RA was examined. In control osteoarthritis patients, these cells exist as a minor population in the peripheral blood. Our data show that in patients with RA, CD57⁺ T cell levels are elevated in peripheral blood, knee joint fluid, knee synovial membrane and bone marrow (BM), compared with peripheral blood of controls. CD57⁺ T cells are especially high in knee joint fluid and joint-adjacent BM, while CD56⁺ T cells show no such increase. CD57⁺ T cells contain a major population of CD8⁺ cells and higher proportions of CD4^?8^? cells and γδ T cells than do CD57^?T cells. CD57⁺T cells in peripheral blood and joint fluid increase with the duration of disease. Erythrocyte sedimentation rate (ESR) is inversely correlated with the proportion of CD57⁺T cells in the joint fluid. Although RA frequently occurrs in patients with CD3⁺57⁺ cell leukaemia, and some CD57⁺T cells are likely to be involved in the onset of RA, we suggest that CD57⁺T cells may rather suppress inflammation of RA, and other cellular components (e. g. granulocytes) may govern the severity of the inflammation of RA. These CD57⁺ T cells are probably generated extrathymically in the adjacent BM or joint space.     

Functional TLR9 modulates bone marrow B cells from rheumatoid arthritis patients

TLR9 recognizes unmethylated CpG‐rich, pathogen‐derived DNA sequences and represents the component of the innate immune system that heavily influences adaptive immunity and may contribute to the immunological disturbances in rheumatoid arthritis (RA). Accumulating data indicate that BM of RA patients participates in the pathogenesis of this disease as a site of proinflammatory cytokines overproduction and lymphocytes activation. Here, we investigated the functionality of TLR9 and its role in the modulation of RA BM B‐cell functions. We report that BM B cells isolated from RA patients express TLR9 at the mRNA and protein levels acquired at the stage of preB/immature B‐cell maturation. Stimulation of BM CD20⁺ B cells by CpG‐containing oligodeoxynucleotide‐enhanced expression of activation markers (CD86 and CD54) triggered IL‐6 and TNF‐α secretion and cell proliferation. Significantly higher levels of eubacterial DNA encoding 16S‐rRNA were found in BM samples from RA than osteoarthritis patients. Moreover, RA BM B cells exerted higher expression of CD86 than their osteoarthritis counterparts, suggesting their in situ activation via TLR9. Thus, our data indicate that TLR9 may participate in direct activation and proliferation of B cells in BM, and therefore could play a role in the pathogenesis of RA.     

Role of CD40 antigen and interleukin-2 in T cell-dependent human B lymphocyte growth

In the present study, we examined the participation of CD40 ligand (L)-CD40 interaction in T cell-dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4⁺ cloned T cells activated with immobilized anti-CD3 antibody. The anti-CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD⁺ B cell was significantly less inhibited by anti-CD40 than that of sIgD^? cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL-2. This contrasts with the CD40 system where B cells are essentially responsive to IL-4 and IL-10 but not to IL-2 alone. Collectively, these data indicate that CD40L-CD40 interaction plays an important role in IL-2-mediated T cell-dependent B cell responses. However, the activation of a subset of sIgD⁺ cells may be independent of this interaction.     

CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4⁺ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8⁺ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8⁺ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8⁺ T cells in BM failure and a beneficial role for Foxp3⁺ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8⁺ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8⁺ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8⁺ T cells dramatically expedite disease progression and promote CD4⁺ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8⁺ T cell (Tc1) response.     

人骨髓间质干细胞向造血细胞分化潜能的实验研究

目的:在体研究人骨髓间质干细胞(hBMMSCs)向造血细胞分化的潜能。方法:将hBMMSCs经尾静脉注射给环磷酰胺处理的严重联合免疫缺陷(SCID)小鼠,利用流式激活细胞分析系统(FACS)检测hBMMSCs输注后存活35d的SCID小鼠外周血、骨髓和脾脏中人源性造血细胞的表型和水平。结果:hBMMSCs输注组外周血(PB)、骨髓(BM)和脾脏(spleen)中可检测到人CD45⁺/H-2D^d-、CD34⁺/H-2D^d-细胞,而对照组PB、BM和脾脏均未检测到上述表型的人造血细胞。结论:hBMMSCs具有向造血细胞分化的潜能。     

Chronic Trichuris muris infection alters hematopoiesis and causes IFN‐γ‐expressing T‐cell accumulation in the mouse bone marrow

Proinflammatory cytokines produced during immune responses to infectious stimuli are well‐characterized to have secondary effects on the function of hematopoietic progenitor cells in the BM. However, these effects on the BM are poorly characterized during chronic infection with intestinal helminth parasites. In this study, we use the Trichuris muris model of infection and show that Th1 cell‐associated, but not acute Th2 cell‐associated, responses to chronic T. muris infection cause a major, transient expansion of CD48^?CD150^? multipotent progenitor cells in the BM that is dependent on the presence of adaptive immune cells and IFN‐γ signaling. Chronic T. muris infection also broadly stimulated proliferation of BM progenitor cells including CD48^?CD150⁺ hematopoietic stem cells. This shift in progenitor activity during chronic T. muris infection correlated with a functional increase in myeloid colony formation in vitro as well as neutrophilia in the BM and peripheral blood. In parallel, we observed an accumulation of CD4⁺, CD8⁺, and CD4^?CD8^? (double negative) T cells that expressed IFN‐γ, displaying activated and central memory‐type phenotypes in the bone marrow during chronic infection. Thus, these results demonstrate that Th1 cell‐driven responses in the intestine during chronic helminth infection potently influence upstream hematopoietic processes in the BM via IFN‐γ.     

Regulatory role of CD95 ligation on human B cells induced in vivo capable of spontaneous and highrate Ig secretion

CD95 ligation elicits apoptotic signals in many cell systems. This study analyzes the effect of anti-CD95 mAb on human cells capable of spontaneous and highrate Ig secretion. Such cells have been induced in vivo and represent a highly mature B cell stage. Addition of the anti-CD95 monoclonal antibody (mAb) CH11 to tonsil B cells inhibited 50–60% of their spontaneous Ig secretion. The effect was exerted early in the culture and could be reversed by a pre-treatment with a neutralizing mAb. N-acetyl-D-sphingosine (C2-ceramide), although not a close analog, also reduced Ig secretion to a similar extent. The inclusion of a tetrapeptide inhibitor for certain interleukin-1β-converting enzyme proteases prevented the inhibitory effect of CH11 mAb on tonsil B cells. B cells capable of spontaneous Ab secretion obtained from blood of recently-immunized volunteers were also inhibited by CH11 mAb and C2-ceramide. In contrast, bone marrow (BM) B cells capable of spontaneous Ig secretion were unaffected by these agents. This CD95 ligation-mediated inhibition of tonsil and blood Ig-secreting B cells could not be reversed by cytokines with demonstrated activity on these B cells. Human mature B cells induced in vivo are identifiable as CD38^hi cells. Flow cytometric analysis revealed that a fraction of tonsil CD38^hi cells expressed low levels of CD95. Moreover, about 20% of these cells exhibited basal apoptosis, as defined by annexin V binding. This phenomenon was markedly increased by CD95 ligation. On the other hand, BM CD38^hi cells showed neither CD95 expression nor CD95-induced annexin V binding. These data suggest that CD95 ligation might play a role in the control of human humoral responses by inducing apoptosis in susceptible mature B cells.     

Normal and clonal B lineage cells can be distinguished by their differential expression of B cell antigens and adhesion molecules in peripheral blood from multiple myeloma (MM) patients—diagnostic and clinical implications

Human MM is a haematologic disorder characterized by the accumulation of malignant plasma cells (PC), primarily in the bone marrow (BM). Although these cells characteristically home to the BM, in recent years several groups have detected the presence of related malignant B cells in the peripheral blood (PB) which could be implicated in the progression and spread of the disease. However, the proportion and origin of these clonotypic circulating B cells is still controversial. In this study, using a triple-staining flow cytometric procedure and a whole blood lysis method, PB B lineage cells could be divided into two populations according to their distinct repertoires of cell adhesion molecules and B cell antigens in untreated MM patients. The results show that: (i) the percentage and the absolute number of PB CD19⁺ B cells were decreased in MM patients compared with controls; (ii) the quantity and percentage of B cell antigens (CD20, CD22, CD24, DR, CD138) and adhesion molecules (β₁- and β₂-integrins, CD44, CD54, CD56, CD61 and CD62L) expressed by these PB CD19⁺ cells of MM patients and healthy subjects were similar and all of them were virtually polyclonal cells; (iii) a very minor circulating CD19^? CD38⁺⁺ CD45^?/dim subset was also detected which expressed CD138 (B-B4) (high intensity), monoclonal cytoplasmic immunoglobulin (cIg), and was negative for pan-B antigens (CD19, CD20, CD24, DR), surface immunoglobulin (sIg) and several adhesion molecules such as CD62L, CD18 and CD11a; this CD19^?CD38⁺⁺ CD45^?/dim CD138⁺⁺ subset was not found in normal blood and exhibited a phenotypic profile which was closely related to that of malignant BM plasma cells, with the exception of the CD56 antigen. Polymerase chain reaction (PCR) analysis of IgH clonotypic rearrangements confirmed these results. We postulate that, in MM patients, circulating B lineage cells may be divided into two different categories: polyclonal CD19⁺ B cells and a very minor proportion of clonal CD138⁺⁺ PC that escape from the BM.     

Human bone marrow contains a subset of quiescent early memory CD8+ T cells characterized by high CD127 expression and efflux capacity

Even today it is still not completely understood how CD8⁺ T‐cell memory is maintained long term. Since bone marrow (BM) is a niche for immunological memory, we sought to identify long‐lasting early memory CD8⁺ T cells in this compartment. To achieve this, we looked for CD8⁺ T cells that are able to efflux Rhodamine 123, a typical property of stem cells. Indeed, we identified a distinct subset of CD8⁺ T cells in BM, with the capacity to efflux and high CD127 expression. These CD127^hi effluxers are conventional CD8⁺ T cells exhibiting a broad TCR‐Vβ repertoire and are generated in response to viral peptides in vitro. CD127^hi effluxer CD8⁺ T cells have an early memory phenotype defined by preferential TNF‐α production and a Bcl‐2^hi, KLRG‐1^low profile. This population has long telomeres and shows constitutively low frequencies of Ki‐67 expression ex vivo, but has a high proliferative and differentiation capacity in vitro. However, IL‐15 downmodulates CD127 in CD127^hi effluxer CD8⁺ T cells in vitro. Consequently, the CD127^low effluxer subset may comprise cells recently exposed to IL‐15. Taken together, CD127^hi effluxer CD8⁺ T cells represent a novel population of early memory T cells resident in BM with properties required for long‐lived memory.     

CXCR4, but not CXCR3, drives CD8+ T‐cell entry into and migration through the murine bone marrow

The BM serves as a blood‐forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T‐cell migration to and localization inside the BM. As BM accumulates many CXCR3‐expressing memory CD8⁺ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8⁺ T cells in BM, is critically important for homing of all CD8⁺ T‐cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8⁺ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM‐1+VCAM‐1+BP‐1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL‐7 and IL‐15. We therefore conclude that CXCR4 is not only crucial for entry of CD8⁺ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.     

Increased frequency of CD56Bright NK‐cells,CD3−CD16+CD56− NK‐cells and activated CD4+T‐cells or B‐cells in parallel with CD4+CDC25High T‐cells control potentially viremia in blood donors with HCV

A detailed phenotypic analysis of major and minor circulating lymphocyte subsets is described in potential blood donors with markers of hepatitis C virus (HCV), including non‐viremic and viremic groups. Although there were no changes in the hematological profile of either group, increased the levels of pre‐NK cells (CD3^?CD16⁺CD56^?) and a lower frequency of mature NK cells (CD3^?CD16⁺CD56⁺) characterized innate immunity in the non‐viremic group. Both non‐viremic and viremic groups displayed significantly increased levels of CD56^Bright NK cells. Furthermore, this subset was significantly elevated in the viremic subgroup with a low viral load. In addition, an increase in the NKT2 subset was observed only in this subgroup. An enhanced frequency of activated CD4⁺ T‐cells (CD4⁺HLA‐DR⁺) was a characteristic feature of the non‐viremic group, whereas elevated CD19⁺ B‐cells and CD19⁺CD86⁺ cell populations were the major phenotypic features of the viremic group, particularly in individuals with a low viral load. Although CD4⁺CD25^High T‐cells were significantly elevated in both the viremic and non‐viremic groups, it was particularly evident in the viremic low viral load subgroup. A parallel increase in CD4⁺CD25^High T‐cells, pre‐NK, and activated CD4⁺ T‐cells was observed in the non‐viremic group, whereas a parallel increase in CD4⁺CD25^High T‐cells and CD19⁺ B‐cells was characteristic of the low viral load subgroup. These findings suggest that CD56^Bright NK cells, together with pre‐NK cells and activated CD4⁺ T‐cells in combination with CD4⁺CD25^High T‐cells, might play an important role in controlling viremia. Elevated CD56^Bright NK cells, B‐cell responses and a T‐regulated immunological profile appeared to be associated with a low viral load. J. Med. Virol. 81:49–59, 2009. © 2008 Wiley‐Liss, Inc.     

Differential Adhesion Molecule Expression on Porcine Mononuclear Cell Populations

Adhesion molecule expression was analysed on porcine blood and lymphoid organ CD4⁺ CD8^? naïve T helper (Th) lymphocytes, CD4⁺ CD8⁺ memory Th lymphocytes (particular to the pig), CD4^? CD8^high cytotoxic T (Tc) lymphocytes, CD4^? CD8^low NK cells (CD3^? in the pig), CD4^? CD8^? T-cell receptor-γδ-positive (TCRγδ⁺) lymphocytes, B lymphocytes and monocytes. While CD44 expression was relatively homogeneous amongst mononuclear cells, differences were noted for the integrins. Blood naïve Th lymphocytes were CD49d^low CD11a^low, as were splenic naïve Th cells; blood memory Th lymphocytes were CD49d^high CD11a^low, splenic memory Th cells were CD49d^high CD11a^high with a CD49d^high CD11a^low subpopulation; blood Tc lymphocytes were mainly CD49d^low CD11a^low, and splenic cells were CD49d^high CD11a^high. Lymph node lymphocytes were more homogeneous in their integrin expression. These were relatively CD49d^low CD11a^low, except the memory Th lymphocytes which had higher integrin expression. B lymphocytes related to the majority of integrin^low T cells, while monocytes and NK cells were CD49d^high CD11a^high; γδ T lymphocytes showed variable CD49d expression but a CD11a^high phenotype. CD49d^high CD11a^high co-expression was found, and this phenotype was typical of, but not exclusive to, CD25⁺ (activated) lymphocytes. These results demonstrated that porcine memory Th lymphocytes and NK cells, as well as activated cells, would have increased integrin-dependent activities compared with naïve Th lymphocytes, and integrin-dependent reactions would probably vary between blood and lymphoid organ cells.     

Adult BM generates CD5+ B1 cells containing abundant N‐region additions

The self‐renewing capacity of B1 cells infers homeostatic regulation; however, previous work suggests the low level of N‐region addition characterizing B1 cells early in life increases with age, which implies that the B1‐cell population is not a closed system. To explore this, we evaluated N‐region addition in CD5⁺ B1 cells generated from adult BM. Adult BM cells were marked with GFP introduced by mouse stem cell virus transduction, and were then adoptively transferred into lethally irradiated recipients. Within 2–3 months, we found GFP‐marked CD5⁺ B cells in the peritoneal cavities of recipients, which we demonstrate here meet a variety of criteria for B1‐cell traits including Mac‐1 surface expression; annexin, elfin, and Pax‐5 gene expression; mitogenic responsiveness to phorbol ester; and spontaneous immunoglobulin secretion. Notably, we found by single‐cell PCR that this population of BM‐derived CD5⁺ B1 cells expressed immunoglobulin with abundant N‐region addition (and little V_H11/V_H12 skewing), unlike CD5⁺ B1 cells obtained from unmanipulated animals but reminiscent of B2 cells. Further, we confirmed that native CD5⁺ B1 cells from older mice contain more N‐region additions than native CD5⁺ B1 cells from younger mice. These results suggest that adult BM progenitors contribute to the peritoneal CD5⁺ B1‐cell pool over time.     

Isolation of peritoneal precursors of B-1 cells in the adult mouse

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220⁺IgM^?lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of λ5, V_preB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220⁺IgM^? cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220⁺IgM^? cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.     

Transitional B cell subsets in human bone marrow

B cells originate from precursors in the bone marrow, and the first cells which migrate to the peripheral blood have been classified as ‘transitional B cells’. Transitional B cells have been characterized in human blood with stage 1 (T1) and stage 2 (T2) subsets being proposed. In the present study, 27 normal human bone marrow samples were analysed for transitional B cell markers by eight‐colour flow cytometry. T1 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD^lo) and T2 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD⁺) were identified in normal bone marrow samples at a mean frequency of 3·2 and 3·1% of total B lineage cells, respectively. A majority of the bone marrow transitional B cells were CD24^hiCD38^hi, the phenotype of blood transitional B cells. Consistent with recent peripheral blood data, T2 B cells had a significantly higher CD21 expression compared with T1 B cells (72·4 versus 40·9%) in the bone marrow. These data raise the possibility that transitional B cells are capable of differentiating from T1 to T2 B cells within the bone marrow. Furthermore, transitional cells at either stages 1 or 2 might be capable of migrating out of the bone marrow.     

Subepithelial B cells in the human palatine tonsil. I. Morphologic,cytochemical and phenotypic characterization

This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5⁻ B cells had a surface phenotype (IgM⁺, IgD⁺, CD23⁻, CD38^±, CD10⁻, CD44⁺) that was different from that of FM (IgM⁺, IgD⁺, CD23⁺, CD39⁺, CD38⁻, CD10⁻, CD44^±) and of germinal center (GC) (CD23⁻, CD39⁻, CD38⁺, CD10⁺, CD44^±, IgG⁺) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5⁻ B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5⁻ B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5⁻ B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5⁻ B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP⁺, while GC cells were consistently ALP⁻. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5⁻ B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5⁻ B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.