B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production

B cell immunoglobulin production is regulated by helper T cells through direct interaction and secreted cytokines. In the present study, we functionally analyzed CD27 in cord and peripheral blood B cells. Adult peripheral blood B cells were separated into CD27⁺ and CD27^? cells, which differed in their morphology. Cord blood B cells did not express CD27, and CD27 expression on peripheral blood B cells increased with age. Only CD27⁺ B cells had the ability to produce immunoglobulin, which was increased by contact with a tumor necrosis factor-related transmembrane ligand, CD70. Adult peripheral blood CD27⁺ B cells can be further subdivided into two discrete subtypes: IgD^?CD27⁺ and IgD⁺ CD27⁺ B cells. IgD^? CD27⁺ B cells produce IgG, IgM and IgA, whereas IgD⁺ CD27⁺ B cells predominantly produce IgM. The addition of activated CD4⁺ CD45RO T cells expressing CD70 caused down-regulation of CD27 expression on activated B cells, and this down-modulation was completely blocked by anti-CD70 monoclonal antibody, indicating direct T-B cell contact via CD27/CD70. The triggering via CD27 and CD40 additively increased the immunoglobulin production under Staphylococcus aureus Cowan strain plus interleukin-2 stimulation. Taken together, our findings demonstrate that peripheral blood B cells are separated into subpopulations by CD27 and IgD expression and that CD27⁺ B cells produce large amounts of immunoglobulin by interaction with the CD70 molecule.     

A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation

Toll‐like receptors (TLRs) recognize specific pathogen‐associated molecular patterns (PAMPs), which subsequently trigger innate immunity. Recent data also suggest a role for TLRs in the direct activation of adaptive immune cells. In the present study, the expression and function of TLR1–TLR10 were characterized in purified human tonsillar B cells, focusing on differences among CD19⁺ CD38^– CD27^– (naïve B cells), CD19⁺ IgD^– CD27^–[germinal centre (GC) B cells] and CD19⁺ CD38^– CD27⁺ (memory B cells) cells. The study was also designed to compare the TLR expression in B cells obtained from infected and hyperplastic tonsils that served as controls. The results demonstrated a distinct repertoire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predominated. No differences were found among naïve, GC and memory B cells. Tonsillar infection did not substantially alter the TLR expression profile in ex vivo‐isolated B‐cell subsets. Purified CD19⁺ B cells stimulated with Pam₃CSK₄, R‐837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin‐6 secretion and an up‐regulated expression of human leucocyte antigen (HLA)‐DR. Collectively, the present study demonstrates that B cells exhibit constitutively high levels of specific TLRs, which are not developmentally regulated during the B‐cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells. Furthermore, the distinct expression of TLRs allows B cells to respond directly to the cognate PAMPs. This further emphasizes the role of TLRs in directly activating adaptive immune cells.     

Phenotype and function of human B cells expressing CD70 (CD27 ligand)

CD70, the cellular ligand of the tumor necrosis factor receptor family member CD27, can be found on a limited number of germinal center (GC) B cells in some tonsils, on scattered lymphocytes residing in secondary lymphoid organs, and on a fraction of the circulating B cell population. Due to the restricted expression of CD70 in vivo, we analyzed signals that determine CD70 expression levels and characterized the phenotype and function of CD70⁺ B cells. Expression of CD70 on B cells activated in vitro was found to be dependent on the continuous presence of a B cell antigen receptor cross-linking agent, and induced or potentiated by CD40 ligation but was down-modulated by the Th2 cytokines interleukin (IL)-4 and IL-13. Both in peripheral blood and tonsil cell suspensions, CD70⁺ B cell subpopulations were found to be enriched for CD27-and IgG-expressing cells, but contained less IgD⁺ B cells. Additional analysis of markers which define specific differentiation stages (Bm1-5) of mature B cells within human tonsils did not place CD70-expressing B cells in one of these subsets. Functional experiments revealed that whereas both CD70⁻ and CD70⁺ B cells can secrete immunoglobulin after activation with a combination of Staphylococcus aureus Cowan strain I and IL-2, only CD70⁺ B cells can produce large quantities of antibodies when stimulated in a T cell-dependent fashion. Our combined data imply that CD70 is a marker for mature B cells which have recently been primed by antigen in vivo.     

Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

CD21–/low B cells in human blood are memory cells

The complement receptor 2 (CR2, CD21) is part of a complex (CD21/CD19/CD81) acting as a co‐receptor to the B cell receptor (BCR). Simultaneous triggering of the BCR and CD21 lowers the threshold for B cell activation. Although CD21 is important, B cells that express low amounts or lack surface CD21 (CD21^–/low) are increased in conditions with chronic inflammation, e.g. autoimmune diseases. However, little is known about the CD21^–/low B cell subset in peripheral blood from healthy donors. Here, we show that CD21^–/low cells represent approximately 5% of B cells in peripheral blood from adults but are barely detectable in cord blood, after excluding transitional B cells. The CD21^–/low subset can be divided into CD38^–24⁺ and CD38^–24^low cells, where most of the CD38^–24⁺ are CD27⁺immunoglobulin (Ig)M⁺IgD⁺ and the CD38^–24^low are switched CD27^–. Expression levels of additional markers, e.g. CD95 and CD62L, are similar to those on classical memory B cells. In contrast to naive cells, the majority of CD21^–/low cells lack expression of the ABCB1 transporter. Stimulation with a combination of BCR, Toll‐like receptor (TLR)?7/8 and interleukin (IL)?2 induces proliferation and differentiation of the CD21^–/low B cells comparable to CD21⁺CD27⁺ memory B cells. The response excluding BCR agonist is not on par with that of classical memory B cells, although clearly above that of naive B cells. This is ascribed to a weaker response by the CD38^–24^low subset, implying that some memory B cells require not only TLR but also BCR triggering. We conclude that the CD21^–/low cells in healthy donors are memory B cells.     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.     

Expression of CD5 and CD38 by human CD5− B cells: Requirement for special stimuli

In this study the mode of expression of CD5 by human tonsillar CD5^? B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5⁺ and CD5^? cells and the two cell fractions exposed to phorbol 12-myristate 13-acetate (PMA). CD5^? B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15–25 % of the total CD5^? B cells were induced to express CD5. Unlike CD5^? B cells, CD5⁺ B cells proliferated vigorously in response to PMA as assessed by [³H] thymidine incorporation and cell cycle analysis in vitro. However, the expression of CD5 by CD5^? B cells was not related to the selective expansion of some CD5⁺ B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5^? B cells remained in the G0-G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [³H] thymidine. Furthermore, mitomycin C treatment of the CD5^? B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5⁺ B cells but not CD5^? B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5⁺ and CD5^? B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5^? B cells. In this respect, the CD5^? B cells that converted into CD5⁺ cells (inducible CD5⁺ B cells) resembled the cells from the CD5⁺ B cell fractions that up-regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin-4 (rIL-4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5^? B cells expressed the CD69 activation marker, no cells other than those co-expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5^? B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL-4-free T cell supernatants. Although this combination of stimuli induced a vigorous cell proliferation, the failure to express CD5 and CD38 was not related to cell cycling, since mitomicyn C-treated CD5^? B cells also failed to express CD5 or CD38 when exposed to PMA in the presence of EL4 cells with or without T cell supernatants. Thus, exposure to T cells alone was sufficient to down-regulate CD5 and CD38 expression. Collectively, the above findings indicate that mature CD5^? B cells can follow distinct pathways of differentiation depending upon the nature of the stimuli encountered, and that CD5 expression may identify a special B cell subset or a particular stage of B cell differentiation.     

TLR9 stimulation drives naïve B cells to proliferate and to attain enhanced antigen presenting function

Mechanisms that regulate naïve B cell proliferation and function are incompletely defined. In this study, we test the hypothesis that naïve B cell expansion, survival and ability to present antigen to T lymphocytes can be directly modulated by Toll‐like receptor (TLR) agonists. In the absence of B cell receptor stimulation, CpG oligonucleotide, a TLR9 agonist, was particularly efficient in inducing naïve B cell proliferation and survival. Although the expanded naïve B cells did not mature into CD27⁺ or IgG⁺ memory B cells, these cells did differentiate into IgM‐secreting cells with increased surface expression of HLA‐DR, CD40 and CD80. This was associated with an increased potential for these B cells to activate allogeneic T cells. We propose that the activation and expansion of naïve B cells induced by TLR9 agonists could enhance the potential of these cells to interact with cognate antigens and facilitate cell‐mediated immune responses.     

TLR4 supports the expansion of FasL+CD5+CD1dhi regulatory B cells,which decreases in contact hypersensitivity

Certain B cells termed as “regulatory B cells” (Bregs) can suppress the ongoing immune responses and a splenic CD5⁺CD1d^hi Breg subset identified earlier was shown to exert its regulatory functions through secretion of IL-10. Though FasL expression is an alternative mechanism of immune suppression used by B cells, little is known about the FasL expressing CD5⁺CD1d^hi Bregs. In this study, we isolated splenocytes or splenic CD19⁺ B cells and compared the efficiency of toll-like receptor(TLR)4 ligand (lipopolysaccharide) with TLR9 ligand (CpG), anti-CD40 and TLR9 ligand (CpG) plus anti-CD40 on the FasL expression of splenic CD5⁺CD1d^hi Bregs by flow cytometry. FasL expression in CD5⁺CD1d^hi B cells was rapidly increased after TLR4 ligation. Intriguingly, anti-CD40 and CpG plus anti-CD40 combinations failed to stimulate FasL expression in CD5⁺CD1d^hi B cells although the IL-10 production was up-regulated in this subset. In addition, LPS and other B10-cell inducers increased the expression of surface molecules like CD86 and CD25, which are correlated to the regulatory functions of B cells. Furthermore, NF-κB and NF-AT inhibitors decreased the TLR4-activated FasL expression in CD5⁺CD1d^hi B cells. Then we sorted splenic CD5⁺CD1d^hi Bregs using flow cytometry and found that TLR4-activated CD5⁺CD1d^hi Bregs suppressed the proliferation of CFSE-labeled CD4⁺ T cells in vitro, which was partly blocked by anti-FasL antibody. In oxazolone-sensitized mice having contact hypersensitivity, FasL expression in splenic CD5⁺CD1d^hi B cells was decreased compared to the control group after TLR4 ligation. Our findings suggest that the regulatory function of CD5⁺CD1d^hi B cells could be partly mediated by Fas-FasL pathway and this FasL expressing CD5⁺CD1d^hi Bregs might participate in the regulation of inflammatory diseases.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

CD27 costimulation contributes substantially to the expansion of functional memory CD8+ T cells after peptide immunization

Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long‐lived memory cells. The costimulatory receptor, CD27, is essential for optimal T‐cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8⁺ T‐cell reactivation remains controversial. We have used OVA and anti‐CD40 to establish a memory CD8⁺ T‐cell population and report here that their secondary expansion, driven by peptide and anti‐CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8⁺ T‐cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector‐ and central‐memory CD8⁺ T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8⁺ T‐cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.     

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.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

Development of CD27+ marginal zone B cells requires GALT

In species other than mouse, little is known about the origin and development of marginal zone (MZ) B cells. Using cross‐reactive antibodies, we identified and characterized splenic MZ B cells in rabbits as CD27⁺CD23^?. In rabbits in which organized gut‐associated lymphoid tissue (GALT) was surgically removed at birth, we found only CD23⁺ follicular (FO) B cells and almost no CD27⁺ MZ B cells in the spleen, indicating that GALT is required for the development of splenic MZ B cells. These findings lead us to suggest that commensal microbiota contribute to the development of MZ B cells.     

The human immunomodulatory CD25+ B cell population belongs to the memory B cell pool

We have shown that human CD20⁺25⁺ B cells display immunomodulatory properties. The aim of this study was to investigate if CD25⁺ B cells are found within the CD27 memory B cell population, and to analyse pattern of their cytokine production. B cells isolated from healthy subjects, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients were analysed regarding the frequency of CD25⁺ B cells within certain B cell subsets. Purified CD25⁺ B cells from healthy subject were used in vitro to evaluate their production of immunomodulatory cytokines. In healthy subjects the majority (60%) of memory B cells (CD20⁺27⁺) also co‐expressed CD25 while only 10–20% of the naïve B cells (CD20⁺27⁻) and plasmablasts (CD20–27⁺) expressed CD25. In RA and SLE patients, we found that 51% and 48%, respectively, co‐expressed CD25 in the memory population, whereas only 11% and 9% co‐expressed CD25 in the naïve B cell population. Phenotypic analysis of the CD20⁺25⁺27⁺ and CD20⁺25⁺27⁻ cells using CD10, CD24, CD38, CD45, CD71, CD80, CD86, CD95, CD138, BAFF‐R, TACI, IgA, IgD, IgG and IgM showed that CD20⁺25⁺27⁺ B cells preferentially represent highly activated, Ig class switched memory B cells. Cytokine profile analysis showed that CD25⁺ B cells secreted significantly higher levels of IL‐10 versus CD25⁻ B cells. In contrast, TGF‐β1 secretion was similar between the CD25⁺ and CD25⁻ sub‐populations. In conclusion, CD20⁺25⁺ B cells constitute a unique subpopulation preferentially occurring among CD20⁺27⁺ memory B cells. We suggest that CD25 can be used as a marker for a memory B cell subset.