Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19(+)/CD27(-) naive B cells more than CD19(+)/CD27(+) memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27(high)/CD38(+)/CD19(dim)/surface Ig(low)/CD20(-)/CD138(+) plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27(high) plasma cells and naive CD27(-) B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27(+) B cells coexpressing IgD were memory B cells preferentially using V(H)3 family members with multiple somatic mutations. CD27(high) plasma cells showed a similar degree of somatic hypermutation, but preferentially employed V(H)4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.     

Intrinsic differences in the proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune responses

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with IL-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division 20-30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38(+) blasts in a manner that increased with consecutive division number. These differentiated CD38(+) B cells divided faster than did CD38(-) memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38(+) and CD38(-) cells present in cultures of memory B cells was increased by IL-10. In contrast, IL-2 enhanced proliferation of CD38(-) and CD38(+) memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.     

B-cell subsets in blood and lymphoid organs in Macaca fascicularis.

BACKGROUND: Cynomolgus monkeys (Macaca fascicularis) are widely used animal models in biomedical research. However, the phenotypic characteristics of cynomolgus monkey (CM) B cells in peripheral blood (PB) and lymphoid organs are poorly understood. METHODS: FACS analyses of PB-, spleen-, lymph node (LN)-, and bone marrow (BM)-derived B cells were performed. RESULTS: CM peripheral blood B cells have a smaller fraction of CD27(-) (naive) cells ( approximately 40%), as compared to human blood samples ( approximately 70%). Similar to humans, an early activation marker, CD23, is expressed more on CD27(-) CM naive B cells, as compared to CD27(+) B cells. The mean fraction of B cells exhibiting a memory phenotype is similar to that seen in human blood. Unlike humans, CM blood contains a subset of CD20(++)CD80(+)CD21(-)IgM(+/-)CD27(+)CD19(+)FSC(++)BAFF-R(low) B cells that are likely of germinal center origin. Thus, CM blood contains (i) a higher percentage of B cells that express the co-stimulatory molecule CD80, and (ii) a lower fraction of B cells that are CD21(+), as compared to human blood. Due to the relative paucity of information on B-cell subsets in organs of healthy humans, a direct comparison between human and CM lymphoid organ data is limited. The fraction of CD27(+) and CD23(+) B cells appears to be similar, while the fraction of CD80(+) B cells appears to be higher than that seen in human lymphoid organs. CM spleens and to some extent lymph nodes have a distinct subset of CD21(++) cells that are also CD80(+/-)CD23(low)IgM(++)CD27(+/-)FSC(++). This subset is phenotypically similar to the marginal zone B cells present in human spleen and LN samples. We also provide detailed analyses on the fraction of lymphoid organ B cells that express CD21, CD23, CD32, and/or CD80 B-cell markers. CONCLUSIONS: In general, cynomolgus monkey B-cell subsets are similar to those seen in humans, as well as to those seen in other nonhuman primates. However, there are some clear differences between human and cynomolgus monkey B-cell subsets. These findings have direct implications for a variety of in vivo studies in cynomolgus monkeys, ranging from basic research on primate B-cell differentiation to models of infectious diseases and trials of new B-cell targeting therapeutic agents.     

IL-27 induces the production of IgG1 by human B cells

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human na?ve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to na?ve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by na?ve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.     

Development and maintenance of a B220- memory B cell compartment

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.     

Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the V(H)1-69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)CD95(-)). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated V(H)1-69(+) B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal V(H)1-69(+) B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.     

Increased CD45RA+ FoxP3(low) regulatory T cells with impaired suppressive function in patients with systemic lupus erythematosus

BACKGROUND: The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4(+)FoxP3(+) T cells into subpopulations during the dynamic SLE development. METHODLOGY/PRINCIPAL FINDINGS: To evaluate the proliferative and suppressive capacities of different CD4(+) T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA(+)FoxP3(low) naive Treg cells (nTreg cells) and CD45RA(-)FoxP3(low) (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA(+)FoxP3(low) nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25(+)CD45RA(+) can be used to defined CD45RA(+)FoxP3(low) nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA(+)FoxP3(low) nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion. CONCLUSIONS/SIGNIFICANCE: Our results indicate that impaired numbers of functional CD45RA(+)FoxP3(low) naive Treg cell and CD45RA(-)FoxP3(low) non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3(+) T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3(+) T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies.     

CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses

High expression of CXCR5 is one of the defining hallmarks of T follicular helper cells (T(FH)), a CD4 Th cell subset that promotes germinal center reactions and the selection and affinity maturation of B cells. CXCR5 is also expressed on 20-25% of peripheral blood human central memory CD4 T cells (T(CM)), although the definitive function of these cells is not fully understood. The constitutive expression of CXCR5 on T(FH) cells and a fraction of circulating T(CM) suggests that CXCR5(+) T(CM) may represent a specialized subset of memory-type T(FH) cells programmed for homing to follicles and providing B cell help. To verify this assumption, we analyzed this cell population and show its specialized function in supporting humoral immune responses. Compared with their CXCR5(-) T(CM) counterparts, CXCR5(+) T(CM) expressed high levels of the chemokine CXCL13 and efficiently induced plasma cell differentiation and Ig secretion. We found that the distinct B cell helper qualities of CXCR5(+) T(CM) were mainly due to high ICOS expression and pronounced responsiveness to ICOS ligand costimulation together with large IL-10 secretion. Furthermore, B cell helper attributes of CXCR5(+) T(CM) were almost exclusively acquired on cognate interaction with B cells, but not with dendritic cells. This implies that a preferential recruitment of circulating CXCR5(+) T(CM) to CXCL13-rich B cell follicles is required for the promotion of a quick and efficient protective secondary humoral immune response. Taken together, we propose that CXCR5(+) T(CM) represent a distinct memory cell subset specialized in supporting Ab-mediated immune responses.     

CD11b expression identifies CD8+CD28+ T lymphocytes with phenotype and function of both naive/memory and effector cells

A previously unreported CD8(+)CD28(+)CD11b(+) T cell subset occurs in healthy individuals and expands in patients suffering from primary viral infections. In functional terms, these cells share the features of naive/memory CD8(+)CD28(+)CD11b(-) and terminally differentiated effector CD8(+)CD28(-)CD11b(+) subpopulations. Like CD28(-) cells, CD28(+)CD11b(+) lymphocytes have the ability to produce IFN-gamma, to express perforin granules in vivo, and to exert a potent cytolytic activity. Moreover, these cells can respond to chemotactic stimuli and can efficiently cross the endothelial barrier. In contrast, like their CD11b(-) counterpart, they still produce IL-2 and retain the ability to proliferate following mitogenic stimuli. The same CD28(+)CD11b(+) subpopulation detected in vivo could be generated by culturing naive CD28(+)CD11b(-) cells in the presence of mitogenic stimuli following the acquisition of a CD45RO(+) memory phenotype. Considering both phenotypic and functional properties, we argue that this subset may therefore constitute an intermediate phenotype in the process of CD8(+) T cell differentiation and that the CD11b marker expression can distinguish between memory- and effector-type T cells in the human CD8(+)CD28(+) T cell subset.     

Kinetics of human B cell behavior and amplification of proliferative responses following stimulation with IL-21

Although recent studies indicated that IL-21 is an important regulator of human B cell activation, detailed comparison of the effects of IL-21 on distinct B cell subsets have not been performed. Our studies revealed that IL-21R is expressed by naive and germinal center B cells, but not memory or plasma cells. IL-21R was increased on naive and memory B cells following in vitro activation. Investigation into the kinetics and magnitude of responses of human B cells to IL-21 revealed that IL-21 potently augmented proliferation of CD40L-stimulated neonatal, splenic naive, and memory and tonsil germinal center B cells. This response exceeded that induced by IL-4, IL-10, and IL-13, cytokines that also induce B cell proliferation. Remarkably, CD40L/IL-21-stimulated naive B cells underwent the same number of divisions as memory cells and exhibited a greater enhancement in their response compared with CD40L alone than memory B cells. Therefore, IL-21 is a powerful growth factor for naive B cells. This may result from the higher expression of IL-21R on naive, compared with memory, B cells. Stimulation of human B cells with CD40L/IL-21 also induced IL-10 production and activation of STAT3. We propose that IL-21 may have therapeutic application in conditions of immunodeficiency where it could expand naive B cells, the predominant B cell subset in such patients. Conversely, because IL-21 is increased in murine models of lupus, dysregulated IL-21 production may contribute to perturbed B cell homeostasis observed in systemic lupus erythematosus. Thus, antagonizing IL-21 may be a novel strategy for treating Ab-mediated autoimmune diseases.     

Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.     

Phenotypic analysis of pneumococcal polysaccharide-specific B cells

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.     

Bm1-Bm5 classification of peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and disturbance in the B cell subpopulations in patients with primary Sjögren's syndrome

Analyses of B cells in the bone marrow and secondary lymphoid tissues have revealed a broad range of cell surface markers defining B cell subpopulations, but only a few of these have been used to analyze B cell subpopulations in peripheral blood (PB). We report here the delineation of circulating PB B cell subpopulations by staining for CD19, CD38, and IgD in combination with CD10, CD44, CD77, CD95, CD23, IgM, and the B cell memory marker CD27. The utility of this approach is shown by the demonstration of disturbances of circulating B cell subpopulations in patients with autoimmune disease. Five mature B cell (Bm) subpopulations were identified in normal PB that were comparable with the tonsillar Bm1, Bm2, early Bm5, Bm5 subpopulations and, surprisingly, to the germinal center (GC) founder cell subpopulation (Bm2' and Bm3delta-4delta), suggesting that some GC founder cells are circulating. No PB B cells resembled the Bm3 and Bm4 GC cells. Remarkably, some cells with the CD38-IgD+ phenotype, previously known as naive Bm1 cells, expressed CD27. The CD38-IgD+ subpopulation therefore includes both naive Bm1 cells and IgD+ memory B cells. This new classification of B cell developmental stages reveals disturbances in the proportions of B cell subpopulations in primary Sj?gren's syndrome (pSS) patients compared with healthy donors and rheumatoid arthritis patients. Patients with pSS contained a significantly higher percentage of B cells in two activated stages, which might reflect a disturbance in B cell trafficking and/or alteration in B cell differentiation. These findings could be of diagnostic significance for pSS.     

EBV persistence involves strict selection of latently infected B cells

EBV is found preferentially in IgD- B cells in the peripheral blood. This has led to the proposal that the recirculating memory B cell pool is the site of long-lived persistent infection. In this paper we have used CD27, a newly identified specific marker for memory B cells, to test this hypothesis. We show that EBV is tightly restricted in its expression. Less than 1 in 1000 of the infected cells in the peripheral blood are naive (IgD+, CD27-) and <1 in 250 are IgD+ memory cells. Furthermore, EBV was undetectable in the self-renewing peripheral CD5+ or B1 cells, a subset that has not been through a germinal center. No such restriction was observed in tonsillar B cells. Therefore, the virus has access to a range of B cell subsets in the lymph nodes but is tightly restricted to a specific long-lived compartment of B cells, the IgD-, CD27+, and CD5- memory B cells, in the periphery. We suggest that access to this compartment is essential to allow the growth-promoting latent genes to be switched off to create a site of persistent infection that is neither pathogenic nor a target for immunosurveillance.     

The microenvironment of human Peyer's patches inhibits the increase in CD38 expression associated with the germinal center reaction

Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer's patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin alpha(4)beta(7), is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-gamma. However, coculture of tonsil naive B cells with an Ab directed against integrin beta(7) inhibits IFN-gamma-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of beta(7) can inhibit the induction of CD38 expression, suggests that ligation of alpha(4)beta(7) in vivo may contribute to a PP-specific GC phenotype.     

Turnover rates of B cells,T cells,and NK cells in simian immunodeficiency virus-infected and uninfected rhesus macaques

We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2'-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4(+) T cells, CD4(-) T cells, CD20(+) B cells, and CD16(+) NK cells in normal uninfected rhesus macaques were 1, 1, 2, and 2%, respectively. Daily turnover rates of CD45RA(-) memory T cells were 1%, and those of CD45RA(+) naive T cells were 0.5% for CD4(+) T cells and approximately 1% for CD4(-)CD45RA(+) T cells. In SIV-infected monkeys with high viral loads, the turnover rates of T cells were increased approximately 2-fold, and that of memory T cells approximately 3-fold. The turnover of CD4(+)CD45RA(+) naive T cells was increased 2-fold, whereas that of CD4(-)CD45RA(+) naive T cells was marginally increased. B cells and NK cells also had increased turnover in SIV-infected macaques, averaging 3 and 2.5% per day, respectively. For all cell types studied here the daily turnover rate increased with the decrease of the CD4 count that accompanied SIV infection. As a consequence, the turnover rates of CD4(+) T cells, CD4(-) T cells, B cells, and NK cells within each monkey are strongly correlated. This suggests that the cellular turnover of different lymphocyte populations is governed by a similar process which one could summarize as "generalized immune activation." Because the viral load and the CD4 T cell count are negatively correlated we cannot determine which of the two plays the most important role in this generalized immune activation.     

Characterization of lymphocyte subsets in patients with common variable immunodeficiency reveals subsets of naive human B cells marked by CD24 expression

Increased proportions of naive B cell subset and B cells defined as CD27(neg)CD21(neg)CD38(neg) are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of κ deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27(neg)CD21(neg)CD38(neg) B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21(pos) naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27(neg)CD21(neg)CD38(neg) B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.     

Loss of IL-7 receptor alpha on CD4+ T cells defines terminally differentiated B cell-helping effector T cells in a B cell-rich lymphoid tissue

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Ralpha (CD127) and the common gamma-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4(+) T cells uniformly express CD127, the memory/effector (non-FOXP3(+)) CD4(+) T cells are divided into CD127(+) and CD127(-) cells. The CD127(-) T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127(+) T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127(-) T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127(+) memory T cells, CD127(-) T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127(-) T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.     

A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus

Human memory B cells comprise isotype-switched and nonswitched cells with both subsets displaying somatic hypermutation. In addition to somatic hypermutation, CD27 expression has also been considered a universal memory B cell marker. We describe a new population of memory B cells containing isotype-switched (IgG and IgA) and IgM-only cells and lacking expression of CD27 and IgD. These cells are present in peripheral blood and tonsils of healthy subjects and display a degree of hypermutation comparable to CD27+ nonswitched memory cells. As conventional memory cells, they proliferate in response to CpG DNA and fail to extrude rhodamine. In contrast to other recently described CD27-negative (CD27neg) memory B cells, they lack expression of FcRH4 and recirculate in the peripheral blood. Although CD27neg memory cells are relatively scarce in healthy subjects, they are substantially increased in systemic lupus erythematosus (SLE) patients in whom they frequently represent a large fraction of all memory B cells. Yet, their frequency is normal in patients with rheumatoid arthritis or chronic hepatitis C. In SLE, an increased frequency of CD27neg memory cells is significantly associated with higher disease activity index, a history of nephritis, and disease-specific autoantibodies (anti-dsDNA, anti-Smith (Sm), anti-ribonucleoprotein (RNP), and 9G4). These findings enhance our understanding of the B cell diversification pathways and provide mechanistic insight into the immunopathogenesis of SLE.