Age‐associated B cells expanded in autoimmune mice are memory cells sharing H‐CDR3‐selected repertoires

Age‐associated B cells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21^?/low). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21^?/low B‐cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus‐prone NZB/W mice and in mice lacking a pre‐B cell receptor (SLC^?/?). However, the nature of the CD21^?/low B cells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC^?/? mice, the vast majority of the ABCs express memory B‐cell (MBC) markers in contrast to wild‐type controls. A similar population is present in lupus‐prone MRL mice before and at disease onset. In SLC^?/? mice, a majority of the ABCs are IgM⁺, their V_H genes have undergone SHM, show clonal diversification and clonal restriction at the H‐CDR3 level. ABC hybridomas, established from SLC^?/? mice, secrete typical lupus autoantibodies, e.g. anti‐Smith antigen, and some of those that bind to DNA comprise a H‐CDR3 that is identical to previously described IgM anti‐DNA antibodies from lupus‐prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H‐CDR3 repertoires.     

IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up-regulation CD80/CD86 co-stimulatory molecules

The expression and function of IgM and IgD antigen receptors were studied in a series of anti-hen egg lysozyme (HEL) immunoglobulin (Ig)-transgenic mice expressing either IgM alone, IgD alone, or both IgM and IgD. B cell surface expression of IgD was found to be more efficient than that of IgM. Thus antigen receptor density on IgD⁺, IgM^? B cells was twofold higher than on IgM⁺, IgD^? B cells despite the presence of sevenfold lower levels of membrane heavy chain mRNA, and coexpression of IgD with IgM led to almost complete inhibition of surface IgM. In addition, less extensive down-regulation of IgD occurred following exposure to antigen in vitro. When regulation of CD80/CD86 co-stimulatory molecules by surface Ig was examined, up-regulation of the former was initiated at lower antigen concentrations on IgM^?, IgD⁺ compared to IgM⁺, IgD^? B cells. On correcting for antigen receptor density, however, induction of CD80/CD86 by IgM and IgD was comparable. Taken together, these results reinforced the functional similarity of IgM and IgD antigen receptors while at the same time revealing differences in expression which may explain their simultaneous presence on mature B cells.     

IL‐10 production in murine IgM+CD138hi cells is driven by Blimp‐1 and downregulated in class‐switched cells

In contrast to antibody‐induced inflammatory responses, some B‐cell subpopulations suppress inflammation through the production of interleukin (IL)‐10. However, the mechanisms underlying Il10 gene expression during B‐cell development is elusive. Here, we identify IgM⁺B220^loCD138^hi cells responsible for marked IL‐10 production in the bone marrow and spleen of mice. These murine IL‐10‐producing cells predominantly secrete IgM and have unique characteristics of long‐lived plasma cells in spite of high expression of surface IgM. We found that IL‐10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp‐1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class‐switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp‐1 expression during the formation of IgM⁺CD138^hi cells without class‐switching elicits IL‐10 production. Adoptive transfer of Il10‐deficient B cells into B‐cell‐deficient mice demonstrated that IgM⁺CD138^hi cell‐derived IL‐10 supports the survival of class‐switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL‐10 secretion by IgM⁺CD138^hi cells in the complete and efficient humoral response.     

CD73 expression identifies a subset of IgM+ antigen‐experienced cells with memory attributes that is T cell and CD40 signalling dependent

B‐cell memory was long characterized as isotype‐switched, somatically mutated and germinal centre (GC)‐derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B‐cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype‐switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T‐dependent versus T‐independent responses. We report that CD73 expression identifies a subset of antigen‐experienced IgM⁺ cells that share attributes of functional B‐cell memory. This subset is reduced in the spleens of T‐cell‐deficient and CD40‐deficient mice and in mixed marrow chimeras made with mutant and wild‐type marrow, the proportion of CD73⁺ IgM memory is restored in the T‐cell‐deficient donor compartment but not in the CD40‐deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age‐associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B‐1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC‐associated memory generation during B‐cell differentiation, CD40‐signalling can influence the composition of the unswitched memory B‐cell pool. They also raise the possibility that a fraction of ABCs may represent T‐cell‐dependent IgM memory.     

Splenic extrafollicular reactions and BM plasma cells sustain IgM response associated with hypercholesterolemia

Hypercholesterolemia associated with atherosclerotic disease is known to be associated with increased total and oxidized (ox) low‐density lipoprotein (LDL)‐specific IgM antibodies in circulation. However, the B‐cell responses accounting for this increase remain to be elucidated. Here, we observed an association between total IgM and oxLDL‐specific IgM autoantibodies with cholesterol in the plasma of hypercholesterolemic apolipoprotein E deficient (apoE^?/?) mice. Our findings also indicated that oxLDL‐specific IgM autoantibodies production was restricted to the spleen, but not the lymph nodes. Further examination of the spleen revealed that the extrafollicular responses, but not germinal center reactions, were the dominant antibody‐producing pathway. A quiescent population of IgM⁺ plasma cells including oxLDL‐specific IgM antibody secreting cells in BM also sustained the elevated IgM antibodies response in circulation. We determined that IgM⁺ plasma cells in the BM were, at least in part, splenic derived by depleting CD11c⁺ DCs and plasmablasts to disrupt the humoral responses. In addition, lowering hypercholesterolemia reduced IgM response by interfering with extrafollicular and BM responses. By elucidating the mechanism underlying the elevated IgM response observed in hypercholesterolemia, this study provides insight into novel immunotherapeutic avenues.     

Unique properties of memory B cells of different isotypes

Summary: Memory antibody responses are typically seen to T-cell-dependent antigens and are characterized by the rapid production of high titers of high-affinity antigen-specific antibody. The hallmark of T-cell-dependent memory B cells is their expression of a somatically mutated, isotype-switched B-cell antigen receptor, features that are mainly generated in germinal centers. Classical studies have focused on isotype-switched memory B cells (mainly IgG isotype) and demonstrated their unique intrinsic properties in terms of localization and responsiveness to antigen re-exposure. However, recent advances in monitoring antigen-experienced B cells have revealed the considerable heterogeneity of memory B cells, which include unswitched IgM⁺ and/or unmutated memory B cells. The IgM and IgG type memory B cells reside in distinct locations and appear to possess distinct origins and effector functions, together orchestrating humoral memory responses.     

T cells recognize the VH complementarity-determining region 3 of the idiotypic protein of B cell non-Hodgkin's lymphoma

The idiotypic protein expressed by B lymphoma cells is a clone-specific tumor antigen which may be suitable for immune targeting by T cells. In this study, we cloned the immunoglobulin heavy chain variable gene (VH) of the idiotypic protein from a patient with B cell lymphoma and used a synthetic peptide of 22 amino acids corresponding to the VH complementarity-determining region (CDR)-3 of the idiotypic protein to investigate whether autologous T cells could recognize this unique peptide. We demonstrated that autologous T cells possessing both CD4⁺ and CD8⁺ phenotypes could be propagated. The T cells were able to proliferate, secrete cytokines, and lyse autologous cells presensitized with the specific peptide in a major histocompatibility complex-dependent manner. Moreover, these CDR3 peptide-primed T cells were also able to kill autologous lymphoma cells. Our results therefore offer new perspectives for specific therapeutic vaccination for the treatment of B cell lymphoma.     

Marginal zone B cells exhibit unique activation,proliferative and immunoglobulin secretory responses

Mouse splenic B cells can be separated based on their distinctive expression of cell surface antigens. Marginal zone (MZ) B cells are CD38^high CD21^high CD23^low/?, while follicular (FO) B cells are CD21^int CD23^int and newly formed (NF) B cells are CD21^dim/? CD23^?. Exploiting these phenotypic distinctions, we isolated the three B cell subsets and assessed their other phenotypic differences and functional capabilities in vitro. FO B cells proliferate more than the other B cell subsets in response to either IgM or CD38 cross-linking. MZ B cells proliferate better than FO B cells when stimulated with lipopolysaccharide (LPS), sub-optimal levels of LPS and CD38 cross-linking or CD40 ligation. When NF, FO and MZ B cells were stimulated with either LPS or LPS and interleukin-4, MZ B cells secreted more IgM and IgG3 than the other two subsets. Similarly, calcium fluxes measured within MZ B cells are larger in amplitude and more sustained after B cell receptor cross-linking than those found in the other two subsets. Collectively, these results indicate that CD38^high CD21^high CD23^low/? MZ B cells are specially suited to play a unique role in response to antigens delivered to the MZ area.     

Generation of precursor,immature, and mature murine B1‐cell lines from c‐myc/bcl‐xL‐overexpressing pre‐BI cells

Deregulated expression of c‐myc and bcl‐xL is long known to generate transformed B cells in humans and mice. We overexpressed these genes to induce in vitro and in vivo differentiation of fetal liver‐derived mouse pre‐BI cells to B1‐lineage pre‐BII‐like, immature and mature B‐cell lines, and to Ig‐secreting cells. In vitro, doxycycline‐controlled c‐myc/bcl‐xL‐overexpressing CD19⁺CD93⁺c‐kikt⁺IgM^? pre‐BI cells differentiate to and survive as CD19⁺CD93⁺c‐kit^?IgM⁺ immature B1 cells. Timed CpG stimulation of these oncogene‐overexpressing pre‐B or immature B1 cells generates either CD19⁺CD93^lowc‐kit^?IgM^?SLC^? pre‐BII‐like or IgM⁺MHCII⁺CD73⁺CD80⁺CD40⁺ mature B1‐cell lines and IgM‐secreting B1 cells in vitro and fixes their state of differentiation. All cell lines are clonable, but a majority of immature and mature B1‐cell clones eventually reach a nonproliferating, surviving G₀‐state. Transplanted in vivo, c‐myc/bcl‐xL‐overexpressing pre‐B cells expand to mature B1 cells, and to IgM‐ and IgA‐secreting plasmablasts and plasma cells. Within 2 months, plasmablasts have expanded most prominently in BM and spleen, indicating that the host selectively expanded development of these transformed plasma cells. The sIgM⁺ B1‐cell lines and clones offer the possibility to study their roles in the development of B1‐Ab repertoires, of B1‐cell‐mediated autoimmune diseases and of B1‐cell malignancies.     

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.     

OX40 controls effector CD4+ T‐cell expansion,not follicular T helper cell generation in acute Listeria infection

To investigate the importance of OX40 signals for physiological CD4⁺ T‐cell responses, an endogenous antigen‐specific population of CD4⁺ T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S‐specific CD4⁺ T cells, but only on a subset that co‐expressed effector cell markers. This population was specifically expanded by Ab‐ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S‐specific effector cells. OX40 was not expressed by 2W1S‐specific memory cells, although it was rapidly up‐regulated upon challenge whereupon Ab‐ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4⁺ T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.     

CD27+ B cells in human lymphatic organs: re-evaluating the splenic marginal zone

The marginal zone of human spleens is regarded as an organ‐specific region harbouring sessile memory B cells. This opinion has arisen by extrapolating from results obtained in mice and rats. Detection of CD27⁺ B cells in situ now revealed similarities among the most superficial region of B‐cell follicles in human spleens, reactive lymph nodes, inflamed appendices, tonsils and terminal ilea. The follicular surface in these organs consists of small naïve immunoglobulin D (IgD)⁺ CD27^– B cells predominating in an inner area and larger IgD^+/– CD27⁺ B cells prevailing in a more superficial position. CD27⁺ B cells may, however, also occupy the entire follicular periphery around the germinal centre. Together with additional peculiarities this distribution indicates a fundamental microanatomical difference among the human and rodent splenic white pulp. We hypothesize that the follicular periphery represents a recirculation compartment both for naïve and memory/natural reactive B cells in all human secondary lymphatic organs. This assumption implies a difference in recirculation behaviour among human and rodent B memory cells.     

Differential expression of CD22 (Lyb8) on murine B cells

Previous studies have established the distribution, biochemistryand functional attributes of human CD22, a B cell-restrictedglycoprotein. Recently, molecular cloning of the murine CD22equivalent revealed this molecule to be the same as the previouslydescribed Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2,the present report documents the expression patterns of CD22within the murine B cell compartment. The results demonstratethat in the bone marrow, murine CD22 is absent on the surfaceof pro-B cells, pre-B cells and newly emerging lgM⁺ B cells.CD22 is present at a low density on immature IgM^hi B cells andfully expressed on mature recirculating B cells. In the periphery,murine CD22 is expressed at mature levels on all B cell subsetsincluding follicular, marginal zone, B1 and switched B cells.Further studies showed CD22 to be retained on activated murineB cells for extended periods. Finally, in combination with CD23and heat stable antigen, CD22 can be used to delineate the immaturesplenic B cells, and distinguish them from follicular and marginalzone cells. Together, the results demonstrate murine CD22 tobe a useful pan marker for all mature B cell subsets.     

Constitutive activation of Bruton's tyrosine kinase induces the formation of autoreactive IgM plasma cells

B‐cell receptor (BCR)‐mediated signals provide the basis for B‐cell differentiation in the BM and subsequently into follicular, marginal zone, or B‐1 B‐cell subsets. We have previously shown that B‐cell‐specific expression of the constitutive active E41K mutant of the BCR‐associated molecule Bruton's tyro s ine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low‐level expression of the E41K or E41K‐Y223F Btk mutants was associated with reduced follicular B‐cell numbers and significantly increased proportions of B‐1 cells in the spleen. Crosses with 3‐83μδ and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B‐1 B‐cell fate choice, but resulted in selective expansion or survival of B‐1 cells. Residual B cells were hyperresponsive and manifested sustained Ca²⁺ mobilization. They were spontaneously driven into germinal center‐independent plasma cell differentiation, as evidenced by increased numbers of IgM⁺ plasma cells in spleen and BM and significantly elevated serum IgM. Because anti‐nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B‐cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B‐cell activation.     

Differential responses of human B-lymphocyte subpopulations to graded levels of CD40-CD154 interaction

Naïve and memory B‐lymphocyte populations are activated by CD154 interaction through cell‐surface CD40. This interaction plays an important role in the regulation of the humoral immune response, and increasing evidence indicates that fine variation in CD40 binding influences B lymphocytes, macrophages and dendritic cells in murine models. Here we have investigated whether and how variations in the intensity of the CD40–CD154 interaction could contribute to differential regulation of human B‐lymphocyte populations. Proliferation and differentiation of B lymphocytes were monitored in response to graded levels of CD40 stimulation in the presence of interleukin (IL)‐2, IL‐4 and IL‐10. Our results show that the level of CD154 binding to CD40 on B lymphocytes can directly influence the evolution of CD19⁺ CD27^– and CD19⁺ CD27⁺ cell populations. Furthermore, proliferation, global expansion of CD19⁺ cells and emergence of CD38⁺⁺ CD138⁺ cells, as well as immunoglobulin G (IgG) and IgM secretion, were affected by the level of exposure of B lymphocytes to CD154. These results suggest that the CD40–CD154 interaction is more like a rheostat than an on/off switch, and its variation of intensity may play a role in the regulation of B‐lymphocyte activation following the primary and/or secondary humoral immune response.     

Antigen Conjugated to Anti‐CD23 Antibodies is Rapidly Transported to Splenic Follicles by Recirculating B Cells

IgE–antigen complexes, administered intravenously to mice, induce a several 100‐fold higher specific antibody response than antigen alone. Additionally, in vivo activation and proliferation of specific CD4⁺ T cells is enhanced. The mechanism behind these effects is thought to be that peripheral B cells capture IgE–antigen complexes via their low‐affinity receptor for IgE, CD23, and rapidly transport them to splenic B cell follicles where an immune response is initiated. Here, we demonstrate that ovalbumin, covalently coupled to anti‐CD23 antibodies and administered intravenously to mice, is also transported to splenic follicles and induces an enhanced primary antibody response. These effects are absent in CD23‐deficient mice. No enhanced induction of immunological memory was observed. These findings extend previous observations regarding the in vivo role of CD23 and emphasize that recirculating B cells play an important role in antigen transport to the spleen.     

Functional Characterization of Murine CD25 Expressing B Cells

B cells are an important part of both innate and adaptive immune system. Their ability to produce antibodies, cytokines and to present antigen makes them a crucial part in defence against pathogens. In this study, we have in naïve Naval Medical Research Institute mice functionally characterized a subpopulation of splenic B cells expressing CD25, which comprise about 1% of the total B cell compartment. Murine spleen cells were sorted into two highly purified B cell populations either CD19⁺ CD25⁺ or CD19⁺ CD25^?. We found that CD25⁺ B cells secreted higher levels of IL‐6, IL‐10 and INFγ in response to different TLR‐agonists, and were better at presenting alloantigen to CD4⁺ T cells. CD25 expressing B cells spontaneously secreted immunoglobulins of IgA, IgG and IgM subclass and had better migratory ability when compared with CD25^? B cells. In conclusion, our results demonstrate that CD25⁺ B cells are highly activated and functionally mature. Therefore, we suggest that this population plays a major role in the immune system and may belong to the memory B‐cell population.     

Human IgM+IgD+ B cells,the major B cell subset in the peripheral blood,express Vϰ genes with no or little somatic mutation throughout life

Peripheral blood B cells of a 67-year-old person were separated into IgM⁺IgD⁺, IgM⁺IgD^?, and IgM^?IgD^? subsets, and nucleotide sequences of expressed immunoglobulin light chain variable (V) regions encoded by V?3 and V?4 gene family members were determined from amplified cDNA. V region sequences from IgM⁺IgD⁺ cells (the major B cell population in the blood) showed no or little somatic mutation (0.3%), in contrast to V? sequences from IgM⁺IgD^? and IgM^?IgD^? B cells (2.0% and 3.9%, respectively). This suggests that in the human like in the mouse, and independently of age, somatically mutated memory B cells accumulate in the compartment of IgM^?IgD^? cells, whereas the IgM⁺IgD⁺ subpopulation consists of cells whose antibody repertoire is mainly determined by V region gene rearrangements and N-region insertion, at the molecular level. The somatically mutated IgM+IgD^? cells may represent early descendants of IgM⁺IgD⁺ cells recruited into the memory cell compartment.