| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Centenary Institute of Cancer Medicine and Cell Biology, and
University of Sydney, Sydney, New South Wales, Australia;
Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia; and
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Ig-secreting cells (ISC) resembling PC can also be generated in vitro by culturing human germinal center or memory B cells with BM stromal cells and/or activated T cells (11, 12), a follicular dendritic-like cell line plus CD40L, IL-2, IL-4, and IL-10 (13, 14) or the T cell-derived stimuli CD40L, IL-2, and IL-10 (15, 16, 17). These cells have typically been identified by the increased expression of CD38, and the acquired ability to secrete high levels of Ig (11, 12, 13, 14, 15, 16, 17). Such cells generated in vitro are rapidly proliferating (17, 18, 19, 20), suggesting they are more akin to plasmablasts detected in vivo (21, 22), rather than mitotically inactive PC (23). By tracking the proliferation history of activated B cells, we have recently found that the differentiation of memory B cells into CD38-expressing cells is IL-10 dependent, and increases in frequency with cell division; that is, CD38+ B cells appear in cultures of memory B cells stimulated with CD40L and IL-10 once the cells have undergone three or more rounds of cell division (17, 20, 24). Furthermore, the survival requirements of the CD38+ B cells differed from CD38– B cells present in the same culture inasmuch that CD38+ B cells became independent of CD40L, and responsive to B-cell activating factor belonging to the TNF family (BAFF), while CD38– B cells required continual stimulation with CD40L for their persistence (11, 17, 25). However, we also found that only 
40% of CD38+ cells secreted Ig, and that there was a population of CD38– ISC (17, 25). Thus, the "PC" phenotype of CD38+ cells generated in vitro did not correlate with PC function. To address this discrepancy, we have now examined in greater detail cultures of activated memory B cells in an attempt to accurately identify the phenotype of ISC generated in vitro. We found that CD27 is up-regulated on the surface of memory B cells in an IL-10-dependent and division-dependent manner, and that ISC segregated into the CD27high subset of activated memory B cells irrespective of the acquired expression of CD38. The CD27high ISC exhibited increased expression of the transcription factors Blimp-1 and X box-binding protein-1 (XBP-1), and reduced expression of Pax-5, as well as phenotypic and migratory characteristics similar to those of human primary PC. In contrast, nondifferentiated B cell blasts (i.e., CD38–CD27+) present in the same culture retained features of memory B cells. These results reveal the dynamic ability of human memory B cells to generate multiple cell fates following activation with T cell-dependent stimuli to yield effector cells, namely ISC, as well as sufficiently expand the pool of nondifferentiated memory B cells to increase the size of this population of effector cells that can then be rapidly activated following re-exposure to the immunizing Ag.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
FITC-conjugated anti-CD20 and PE-conjugated anti-CD19 and CD45 mAb were purchased from BD Biosciences; anti-CCR7, PE-conjugated isotype controls, CD21, CD27, CD80, CD86, CD95, and CXCR4 mAb, biotinylated anti-CD44, anti-hamster IgG, and streptavidin (SA)-conjugated to PerCp were purchased from BD Pharmingen. PE-anti-CD22, CD23, CD31, CD38, CD62L, CD138, HLA-DR mAb, biotinylated IgG1 isotype control, and anti-CD38 mAb, allophycocyanin-conjugated isotype control, anti-CD20 and anti-CD38 mAb, and SA-allophycocyanin were purchased from Caltag Laboratories; biotinylated anti-CXCR5 mAb was from R&D Systems; biotinylated anti-human CD27 mAb was from eBioscience; PE-conjugated anti-CD40 mAb (mAb89) was provided by J. Banchereau (Schering Plough Labs, Dardilly, France); PE-conjugated anti-CD39 (A1) mAb has been described previously (26). Purified and biotinylated F(ab')2 of goat anti-human IgM, IgG, or IgA polyclonal Ab were purchased from Southern Biotechnology. Recombinant human BAFF, and mouse anti-BAFF receptor (anti-BAFF-R; clone 9.1), anti-transmembrane activator of and CAML interactor (TACI; clone C4D7) and hamster anti-B cell maturation Ag (BCMA; clone C4E2.2) mAbs were provided by Dr. S. Kalled (Biogen Idec, Cambridge, MA; Ref. 25). Recombinant CD40L expressed as membranes in Sf21 insect cells infected with baculovirus vector containing human CD40L cDNA was provided by Dr. M. Kehry (Boehringer Ingleheim, Ridgefield, CT). IL-2 was purchased from Endogen; IL-10 was provided by Dr. R. de Waal Malefyt (DNAX Research Institute, Palo Alto, CA). CFSE was from Molecular Probes. Recombinant human CXCL12 and CCL21 were purchased from PeproTech; CXCL13 was from R&D Systems.
Generation of CD70 transfectants
A complementary DNA encoding human CD70 was amplified from RNA prepared from the CD70-expressing human B cell line JY by PCR using Pfu polymerase and the following primers (Sigma-Genosys): 5'-GCA TGC GGA TCC TTC CTT CCT TCT CGG CAG CG (BamH1 site underlined), and 3'-GCA TGC GCG GCC GCA ATC AGC AGC AGT GGT CAG GG (NotI site underlined). The resulting product was digested with BamH1 and NotI, and ligated into pcdef3, a derivative of pEF-BOS containing the neomycin-resistance gene (27). The mouse mastocytoma cell line P815 was transfected with CD70/pcdef3 by electroporation, and positive cells were selected initially in the presence of G418 (Bio-Rad), and subsequently by cell sorting.
Cells
Total human B cells (>98% CD19+) were isolated as previously described (17, 28). Memory B cells were isolated by sorting on a FACStarPLUS or FACSVantage (BD Biosciences) following labeling with FITC-anti-CD20 and PE-anti-CD27 mAb and collecting CD27+CD20+ B cells (17, 26).
CFSE labeling and B cell cultures
Primary cultures. Memory B cells were labeled with CFSE (29) and cultured in 48-well plates (4 x 105/ml; BD Labware) for 5 days with CD40L alone (at a predetermined optimal dilution of the membrane preparation; 1/250), or in the presence of IL-10 (100 U/ml), or IL-2 (50 U/ml) plus IL-10. The cells were harvested and expression of CD27 and CD38 was then determined by immunofluorescence and flow cytometric analysis.
Secondary cultures.
CFSE-labeled memory B cells were cultured for 4 days with CD40L, IL-2, and IL-10. The cells were harvested, washed, and recultured (2 x 105/ml) with IL-2 and IL-10 in the absence or presence of CD40L (1/500 dilution) or BAFF (2.5 µg/ml) for an additional 4 days (15, 17, 25). In some experiments, the activated memory B cells were recultured with parental P815 or CD70/P815 cells in the presence of IL-2 and IL-10. Before culture, the P815 cells were fixed in 1% formaldehyde, prepared in PBS, for 20 min (30), washed three times with PBS, and cultured in medium for 1 h to remove the formaldehyde. The memory B cells and P815 cells were cultured at a ratio of 5:1.
At the completion of the primary or secondary cultures, a known number of CaliBRITE beads (BD Biosciences) were added to culture wells before harvesting, and the number of viable B cells was calculated as a function of the ratio of beads to live cells (29). All cultures were performed using RPMI 1640 containing L-glutamine (Invitrogen Life Technologies), 10% FCS (CSL), 10 mM HEPES (pH 7.4) (Sigma-Aldrich), 0.1 mM nonessential amino acid solution (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich), 100 µg/ml Normocin (InVivoGen), and 40 µg/ml apo-transferrin (Sigma-Aldrich) and were conducted at 37°C in a humidified atmosphere containing 5% CO2.
Immunofluorescent staining
For phenotypic analysis, cells were incubated on ice with PE-, allophycocyanin-, or biotinylated specific mAb, or the appropriate isotype control, followed by SA-PerCp and analyzed on a FACSCalibur using CellQuest software (BD Biosciences). Binding of BAFF to activated B cells, as well as assessment of expression of the different BAFF receptors (i.e., BAFF-R, TACF, and BCMA) were determined as described previously (25)
Analysis of Ig secretion
ELISPOT. CFSE-labeled memory B cells were cultured with CD40L, IL-2, and IL-10 for 4 days and then washed and recultured under the same conditions for a further 4 days. The cells were harvested and labeled with PE-anti-CD27 mAb and allophycocyanin-anti-CD38 mAb. Different populations of activated B cells, defined by division history and surface phenotype, were sorted directly into ELISPOT plates (Multiscreen-HA plates; Millipore), and the frequency of cells secreting IgM, IgG, and IgA was determined (17).
ELISA.
CFSE-labeled memory B cells were cultured for 5 days with CD40L, IL-2, and IL-10 and then labeled with PE-anti-CD27 and allophycocyanin-anti-CD38 mAbs. Different populations of activated B cells were isolated by cell sorting and then recultured (105 cells/500 µl/well) for a further 2 days with CD40L, IL-2, and IL-10, after which time supernatants were collected and the level of secreted Ig was determined (17).
Analysis of expression of transcription factors
Semiquantitative PCR was used to examine gene expression in different populations of activated human memory B cells. B cell subsets were isolated by cell sorting, total RNA was extracted (Qiagen RNeasy Kit; Qiagen) and then transcribed into cDNA using oligo-dT (Boehringer Mannheim) or random hexamers (Invitrogen Life Technologies) as primer and Superscript II RNase H– reverse transcriptase (Invitrogen Life Technologies). Resulting cDNA was then normalized for expression of the constitutively expressed housekeeping gene GAPDH (5'-CCA CCC ATG GCA AAT TCC ATG GCA, 3'-TCT AGA CGG CAG GTC AGG TCC ACC) and then used as a template for PCR using REDTaq (Sigma-Aldrich) (3). The following primers were used (Sigma-Genosys): Pax-5 5'-GCA TAG TGT CCA CTG GCT CC; Pax-5 3'-CCA GGA GTC GTT GTA CGA GG; BLIMP-1 5'-GAT GCG GAT ATG ACT CTG TGG; BLIMP-1 3'-CTC GGT TGC TTT AGA CTG CTC; XBP-1 5'-GCT CAG ACT GCC AGA GAT CG; XBP-1 3'-GTC CSG AAT GCC CAA CAG G; Bcl-6 5'-CTG ACA GCT GTA TCC AGT TCA CC; Bcl-6 3'-TCT TGG GGC ATC AGC ATC.
Expression of Blimp-1 and Pax-5 protein by activated human memory B cells was also determined. B cell subsets were isolated by cell sorting, and then lysed in ice cold lysis buffer (10 mM Tris-HCl (pH 7.8), 1% Nonidet P-40, 150 mM NaCl, and enzyme inhibitors). Cell lysates were electrophoresed through 12% acrylamide gels containing 0.1% SDS and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed with Abs against Blimp-1 (31), Pax-5 (C-20; Santa Cruz Biotechnology) or SHP-2 (Santa Cruz Biotechnology) followed by HRP-conjugated anti-rat, anti-goat, and anti-rabbit Ig antiserum, respectively (all from Santa Cruz Biotechnology). The membranes were developed using ECL (Pierce) and autoradiography.
Chemotaxis assays
Migration assays of activated memory B cells were performed using 5-µm Costar Transwell plates (Corning). CXCL12 (100 ng/ml), CXCL13 (1000 ng/ml), or CCL21 (600 ng/ml) (32) were diluted in RPMI 1640 containing 0.5% BSA (chemotaxis medium) and added to wells of a 24-well plate in 600 µl. Chemotaxis medium was used as a control for basal cell migration. CFSE-labeled memory B cells were activated with CD40L, IL-2, and IL-10 for two lots of 4 days, then were washed, and were resuspended in chemotaxis medium, and 5 x 105 cells were added to the upper chamber of the Transwell in 100 µl. Plates were incubated for 4 h at 37°C. A known number of Calibrite beads was then added to each bottom well before harvesting, and the number of B cells that had migrated was calculated. The migrated population was stained with anti-CD27 and CD38 mAb to determine the proportions of different populations of activated memory B cells which were then used to calculate the absolute number of cells of each population that had migrated. Migration was calculated as the percentage of input cells.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Human PC express a higher level of CD27 than memory B cells (3, 6, 9, 14, 33). Therefore, it was of interest to investigate the expression of CD27 on memory B cells cultured under conditions that induce their proliferation and differentiation to CD38+ B cells, which contain a population of ISC. Expression of CD27 on CD40L-stimulated memory B cells did not greatly change with division (Fig. 1a, left panel), remaining at a level comparable to cells before culture (data not shown). Addition of IL-10 alone or in combination with IL-2 resulted in the appearance of a population of cells expressing an increased level of CD27 (Fig. 1a, middle and right panels). Notably, this population increased in frequency with cell division in a manner analogous to the appearance of the CD38+ cells (Fig. 1, a and b).
|
Proliferating B cells expressing increased levels of CD27 are enriched for ISC
The ability of distinct populations of activated B cells, defined by division history and differential expression of CD27 and CD38, to produce Ig was next determined. For these studies, B cells were initially resolved into populations that had undergone the following: <3 divisions (population 1); several rounds of division and remained CD38– (population 2); or undergone the same number of cell divisions as population 2 yet had differentiated to become CD38+ (population 3; see Fig. 1b; Refs. 17 and 25). ISC increased in frequency in populations 2 (CD38–) and 3 (CD38+) of activated memory B cells, compared with population 1 (Fig. 2a) (17). However, these populations were heterogeneous as demonstrated by <40% of them secreting IgM, IgG, and IgA (Fig. 2a). Further heterogeneity was apparent from the finding that within the CD38– and CD38+ populations in the later divisions, there were cells that expressed different levels of CD27 (i.e., low or high; Fig. 1c). To determine whether the ISC in populations 2 and 3 were partitioned into subsets expressing different levels of CD27, population 1, population 2 CD27low, population 2 CD27high, population 3 CD27low, or population 3 CD27high were examined by ELISPOT. For both populations 2 and population 3, ISC were enriched in the CD27high subset, containing up to 5-fold more ISC than the CD27low subset (Fig. 2b). This analysis revealed that up to 80% of these cells were ISC, an 2-fold increase over the total population 2 or 3 (compare Fig. 2, a and b).
|
Phenotypic characterization of plasmablasts generated in vitro
In addition to increasing expression of CD38 and CD27, human PC alter the expression of other molecules such that their phenotype is distinct from that of naive or memory B cells. Compared with mature B cells, primary PC down-regulate CD20, CD21, CD22, CD84, BAFF-R, CXCR5, CCR7, and, to a lesser extent, CD19, CD40, and HLA-DR (3, 32). In contrast, expression of CD31, CD39, CD44, CD49d, CD86, and CD95 is increased while CD45 and CXCR4 remain unchanged (3, 32). Similarly, BM PC acquire expression of CD138 (5). To extend the characterization of ISC generated from memory B cells in vitro, we determined the phenotype of activated cells corresponding to population 1, and populations 2 and 3 that were either CD27low or CD27high. The phenotype of the ISC (i.e., CD27high populations 2 and 3) bore a striking resemblance to each other as well as to in vivo-derived PC inasmuch that expression of CD19, CD20, CD21, CD22, CD40, CD84, CXCR5, HLA-DR, and BAFF-R were reduced compared with the nonsecreting cell in population 1 as well as the CD27low subsets of populations 2 and 3 (Fig. 3; Table I). Similarly, CD39, CD95 (Table I), CD86, CD44, CD49d, and CD62L (Fig. 3) were uniformly expressed on CD27high ISC in populations 2 and 3 while expression of these molecules on the CD27low B cells was heterogeneous, with bimodal expression of some molecules being detected. CD31 and BCMA were weakly induced on activated B cells in populations 2 and 3, while expression of CD45 and CXCR4 was similar on all populations (Fig. 3, Table I), consistent with the phenotype of primary PC (3, 5, 32). No induction of CD138 expression was detected on the activated splenic memory B cells (Table I), analogous to its absence from primary PC in human spleen (3) and some in peripheral blood and tonsil (5). Collectively, the phenotypes of CD27high cells in populations 2 and 3 are very similar, consistent with the functional similarities of these cells (Fig. 2b). Remarkably, the phenotype of population 1 B cells was similar to resting human memory B cells (Fig. 3, Table I; Refs. 3 and 26), suggesting that these cells remained nondifferentiated with the potential to expand the memory B cell pool.
|
|
Differentiation of mature B cells into ISC is accompanied by changes in expression of specific transcription factors. For instance, while Pax-5 is expressed by naive and memory B cells, its expression is extinguished in PC. Similarly, expression of Bcl-6 is restricted to germinal center B cells (34) while Blimp-1 and XBP-1 are induced at the PC stage (3, 23, 35, 36, 37). Therefore, expression of these transcription factors during the in vitro differentiation process that yields ISC from memory B cells was assessed. Analysis of activated memory B cells revealed that expression of Pax-5 was maintained in population 1 (Fig. 4a, lane 2), but decreased in cells that had undergone further proliferation (Fig. 4a, lane 3–6). Interestingly, while Pax-5 was absent from population 3 CD27high B cells, it remained detectable in the CD27low subset of population 3 (Fig. 4a, lane 5). It is possible that the heightened expression of Pax-5 by these cells prevents them from becoming efficient ISC (Fig. 2), because Pax-5 may counter the inductive effect of XBP-1 on the differentiation of these cells toward an ISC fate (23). In contrast to Pax-5, Blimp-1 (Fig. 4b) and XBP-1 (Fig. 4c) were low in population 1 (lane 2) and incrementally increased as the B cells underwent proliferation and differentiation to an effector ISC (lanes 3–6), such that CD27high cells within population 3 expressed the highest levels of these genes. Bcl-6 was induced in activated B cells, down-regulated in population 2 and again increased as the cells acquired expression of CD38 to become population 3 (Fig. 4d).
|
Effect of BAFF and CD70 on the generation of different populations of activated memory B cells
The number of B cells in populations 2 and 3 can be influenced by CD40L or BAFF in the secondary cultures. CD40L is critical for the persistence of population 2 B cells, and can increase population 3 B cells, while BAFF preferentially favors the survival of population 3 (17, 25). Interestingly, culture of activated memory B cells with CD27 ligand (CD70), another TNF superfamily molecule, can increase the proportion of CD38+ B cells generated from memory B cells (30). However, the effect of CD70 on the number of surviving B cells, or different subsets of activated B cells, has not been examined. Therefore, we investigated whether the different B cell populations preferentially responded to secondary stimulation through different BAFF receptors or CD27.
BAFF increased the survival of population 2 CD27high B cells, as well as both subsets of population 3, yet had no significant effect on population 1 nor the CD27low subset of population 2 (Fig. 5a). However, the greatest effect of BAFF was on population 3 CD27high B cells, where almost 5-fold more cells were generated compared with secondary cultures containing IL-2 and IL-10 alone (Fig. 5a). Consequently, in the presence of BAFF, this population of cells dominated the culture, comprising >50% of total cells, compared with cultures containing IL-2 and IL-10 alone, where the CD38+CD27high cells represented <30%. When activated memory B cells were cultured with CD70 transfectants, there was an increase in the number of population 1 B cells, as well as the CD27high cells in populations 2 and 3 (Fig. 5b). Consistent with the down-regulation in CD27 expression, there was a lesser effect on the CD27low B cells (Fig. 5b). The ability of CD70-expressing transfectants to increase the number of CD27high B cells (i.e., ISC) correlated with increased Ig production in these cultures (data not shown). Thus, both BAFF and CD70 are capable of increasing the generation of ISC from memory B cells.
|
A characteristic of B cell development and differentiation is the alteration in responsiveness to chemokines. For instance, B cells increase responsiveness to the lymphoid chemokines CXCL12, CXCL13, and CCR7 ligands (CCL19, 21) as they develop from pro-B cells in the BM through to follicular B cells in the spleen (40). In contrast, PC lose responsiveness to CXCL13 and CCR7 ligands, yet retain the ability to respond to CXCL12 (32, 41, 42). Given the alteration in expression of receptors for these chemokines on the different populations of activated memory B cells, the chemotactic responses of these populations was next examined. Population 1 B cells exhibited chemotactic responses to CXCL12, CXCL13, and CCL21 (Fig. 6). As the activated memory B cells underwent proliferation and differentiation to ISC, their responsiveness to CXCL13 and CCL21 gradually declined such that population 3 CD27high B cells were almost unresponsive to these chemotactic ligands (Fig. 6). In contrast, activated B cells continued to respond to CXCL12, with an increased response being evident for population 3 CD27high B cells (Fig. 6). Thus, in vitro-generated ISC behave analogously to primary PC with respect to chemotactic responses.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Another novel finding in our study was the identification of a CD38–CD27high population with a phenotype and capacity to secrete Ig that was identical with the CD38+CD27high B cells (Figs. 2 and 3). Despite these similarities, some important differences were found between these two populations of in vitro-derived ISC. First was their responsiveness to BAFF. This current study refined our previous analysis (25) by revealing that the greatest effect of BAFF was on the viability of CD38+CD27high ISC, while its effects on CD38–CD27high ISC were modest (Figs. 5 and 7). Second, CD38+ B cells proliferate at a greater rate than CD38– B cells (17, 20, 25), revealing CD38+CD27high ISC to be plasmablasts. A molecular explanation for this difference may be attributable to differential expression of Bcl-6. CD38– B cells (Population 2) expressed less Bcl-6 than CD38+ B cells (population 3; Fig. 4). Interestingly, Bcl-6 can regulate the cell cycle by controlling expression of genes such as c-myc (46). This is further exemplified by the finding that the frequency of proliferating cells in Bcl-6 transgenic mice is markedly enhanced compared with control mice (47), and that overexpression of dominant-negative Bcl-6 arrested proliferation of a human B cell line (46). Thus, increased expression of Bcl-6 by CD38+ B cells may provide them with the molecular machinery required for differentiation into rapidly dividing plasmablasts. An additional explanation for the differences between the CD38–CD27high and CD38+CD27high ISC may be that these cells arise from different progenitor memory B cells, namely IgM-expressing or Ig isotype switched memory cells (24, 28). The finding that 1) CD38–CD27high ISC produced a greater proportion of IgM than CD38+CD27high ISC (Fig. 2), 2) BAFF preferentially induces Ig secretion by isotype switched memory B cells (25), and 3) a greater frequency of isotype switched B cells enter their first cell division than IgM-memory B cells (20) would be consistent with the proposal that IgM-memory B cells preferentially yields CD38–CD27high ISC while CD38+CD27high ISC arise from switched memory B cells.
The coexpression of Blimp-1 and Bcl-6 by B cells that have differentiated to become ISC (CD27highCD38+; Fig. 4) was notable because these transcription factors cross-regulate the expression and function of each other (23, 46, 48). Kallies et al. (31) recently described the generation of a mouse line where GFP was introduced into the Blimp-1 locus. This study elegantly demonstrated that all GFP+ cells were ISC, irrespective of surface phenotype. Interestingly, the stage of PC maturation could be inferred from the relative expression of Blimp-1, with cells expressing intermediate levels of Blimp-1 (i.e., Blimp-1/GFPint) representing immature PC while Blimp-1/GFPhigh cells were mature PC (31). Importantly, a population of Blimp-1/GFPint cells also expressed Bcl-6 and were rapidly dividing, leading to the suggestion that they are plasmblasts (31). These data are consistent with our proposal that CD38+CD27high cells, that express Blimp-1 and Bcl-6, are plasmblasts (Fig. 7), and most likely represent the human counterpart of the murine Blimp-1/GFPint cells (31). Another recent study also found that murine B cells could be induced to differentiate into ISC that expressed both Bcl-6 and Blimp-1, but not Pax-5, when stimulated with IL-21 (49). Thus, because both IL-21 and IL-10 are potent growth and differentiation factors for human (IL-10, IL-21; Refs. 50, 51, 52) and murine B cells (IL-21 only; Refs. 49 and 51), it is likely that the molecular alterations induced by these cytokines are similar.
Our examination also identified two other populations of B cells that appear to have distinct functions and fates: the CD38–CD27low and CD38+CD27low cells that were nonsecreting cells. Interestingly, only 50% of PC identified in human or murine lymphoid tissues by a CD38++CD20± or CD138+B220± phenotype, respectively, cells secrete Ig (3, 53). Thus, it is possible that the CD38+CD27low nonsecreters in vitro (Fig. 2b) correspond to these cells in vivo. Alternatively, CD38+CD27low cells may contain precursors of CD38+CD27high cells and could therefore undergo further differentiation to this phenotype and function (Fig. 7). This indeed appears to occur because CD38+CD27low cells can yield CD38+CD27high cells, as well as produce large amounts of Ig, following isolation and subsequent culture (not shown). Similarly, the CD38–CD27low cells may correspond to a pool of memory B cell precursors that replenish the memory B cell population to prevent depletion of these cells following their differentiation to ISC (53, 54).
Our finding of a CD38– population of ISC suggests the existence of an analogous population in vivo. Indeed, it has been reported that 60–80% of human ISC expressed CD38 (55, 56, 57). More recently, a CD38– population of ISC, that appeared to be the precursor of CD38+ ISC, was identified in human tonsils (44). These CD38– ISC expressed CD19, CD27, CD45, HLA-DR, and Blimp-1 (44), analogous to ISC generated in vitro (Fig. 3, Table I) and in vivo (3, 4, 5, 7). Thus, CD38–CD27high ISC generated in vitro from human splenic memory B cells may correspond to the 20–40% of ISC detected ex vivo that were also CD38–. Our findings regarding human ISC are also consistent with the recent descriptions of murine PC that lack CD138 (31, 58). Taken together, these studies reveal the vast heterogeneity in different populations of ISC both in vivo and in vitro and infer that, in the absence of a definitive cell surface marker, ISC cannot by solely defined by phenotype. We propose that human ISC can be resolved by increased expression of CD27, while the coexpression of CD38 delineates the ISC population into an early stage of commitment to the ISC lineage (CD38–) and plasmablasts (CD38+) (Fig. 7). These findings will further our understanding of the complex regulation of the pathways B cells undergo during differentiation to an effector cell.
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
1 This work was supported by the National Health and Medical Research Council (NHMRC) of Australia. S.G.T. is the recipient of an RD Wright Biomedical Career Development Award from the NHMRC; P.D.H. is a Principal Research Fellow of the NHMRC; L.M.C. is a Senior Research Fellow of the NHMRC. 
2 Address correspondence and reprint requests to Dr. Stuart Tangye, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown 2042, New South Wales, Australia. E-mail address: s.tangye{at}centenary.usyd.edu.au
3 Abbreviations used in this paper: PC, plasma cell; BM, bone marrow; ISC, Ig-secreting cell; SA, streptavidin; BAFF, B-cell activating factor belonging to the TNF family; BAFF-R, BAFF receptor; BCMA, B cell maturation Ag; TACI, transmembrane activator of and CAML interactor; XBP-1, X box-binding protein-1.
Received for publication October 25, 2004. Accepted for publication January 19, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Borte, Q. Pan-Hammarstrom, C. Liu, U. Sack, M. Borte, U. Wagner, D. Graf, and L. Hammarstrom Interleukin-21 restores immunoglobulin production ex vivo in patients with common variable immunodeficiency and selective IgA deficiency Blood, November 5, 2009; 114(19): 4089 - 4098. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. Henn, J. Rebhahn, M. A. Brown, A. J. Murphy, M. N. Coca, O. Hyrien, T. Pellegrin, T. Mosmann, and M. S. Zand Modulation of Single-Cell IgG Secretion Frequency and Rates in Human Memory B Cells by CpG DNA, CD40L, IL-21, and Cell Division J. Immunol., September 1, 2009; 183(5): 3177 - 3187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hauser, J. Verma-Gaur, A. Wallenius, and T. Grundstrom Initiation of Antigen Receptor-Dependent Differentiation into Plasma Cells by Calmodulin Inhibition of E2A J. Immunol., July 15, 2009; 183(2): 1179 - 1187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sarantopoulos, K. E. Stevenson, H. T. Kim, C. S. Cutler, N. S. Bhuiya, M. Schowalter, V. T. Ho, E. P. Alyea, J. Koreth, B. R. Blazar, et al. Altered B-cell homeostasis and excess BAFF in human chronic graft-versus-host disease Blood, April 16, 2009; 113(16): 3865 - 3874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Douagi, C. Gujer, C. Sundling, W. C. Adams, A. Smed-Sorensen, R. A. Seder, G. B. Karlsson Hedestam, and K. Lore Human B Cell Responses to TLR Ligands Are Differentially Modulated by Myeloid and Plasmacytoid Dendritic Cells J. Immunol., February 15, 2009; 182(4): 1991 - 2001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Doria-Rose, R. M. Klein, M. M. Manion, S. O'Dell, A. Phogat, B. Chakrabarti, C. W. Hallahan, S. A. Migueles, J. Wrammert, R. Ahmed, et al. Frequency and Phenotype of Human Immunodeficiency Virus Envelope-Specific B Cells from Patients with Broadly Cross-Neutralizing Antibodies J. Virol., January 1, 2009; 83(1): 188 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tabera, J. A. Perez-Simon, M. Diez-Campelo, L. I. Sanchez-Abarca, B. Blanco, A. Lopez, A. Benito, E. Ocio, F. M. Sanchez-Guijo, C. Canizo, et al. The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes Haematologica, September 1, 2008; 93(9): 1301 - 1309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Diehl, H. Schmidlin, M. Nagasawa, S. D. van Haren, M. J. Kwakkenbos, E. Yasuda, T. Beaumont, F. A. Scheeren, and H. Spits STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation J. Immunol., April 1, 2008; 180(7): 4805 - 4815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Capolunghi, S. Cascioli, E. Giorda, M. M. Rosado, A. Plebani, C. Auriti, G. Seganti, R. Zuntini, S. Ferrari, M. Cagliuso, et al. CpG Drives Human Transitional B Cells to Terminal Differentiation and Production of Natural Antibodies J. Immunol., January 15, 2008; 180(2): 800 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. L. Bryant, C. S. Ma, D. T. Avery, Y. Li, K. L. Good, L. M. Corcoran, R. de Waal Malefyt, and S. G. Tangye Cytokine-Mediated Regulation of Human B Cell Differentiation into Ig-Secreting Cells: Predominant Role of IL-21 Produced by CXCR5+ T Follicular Helper Cells J. Immunol., December 15, 2007; 179(12): 8180 - 8190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Darce, B. K. Arendt, X. Wu, and D. F. Jelinek Regulated Expression of BAFF-Binding Receptors during Human B Cell Differentiation J. Immunol., December 1, 2007; 179(11): 7276 - 7286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wei, J. Anolik, A. Cappione, B. Zheng, A. Pugh-Bernard, J. Brooks, E.-H. Lee, E. C. B. Milner, and I. Sanz A New Population of Cells Lacking Expression of CD27 Represents a Notable Component of the B Cell Memory Compartment in Systemic Lupus Erythematosus J. Immunol., May 15, 2007; 178(10): 6624 - 6633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Darce, B. K. Arendt, S. K. Chang, and D. F. Jelinek Divergent Effects of BAFF on Human Memory B Cell Differentiation into Ig-Secreting Cells J. Immunol., May 1, 2007; 178(9): 5612 - 5622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Konforte and C. J. Paige Identification of Cellular Intermediates and Molecular Pathways Induced by IL-21 in Human B Cells J. Immunol., December 15, 2006; 177(12): 8381 - 8392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Racanelli, M. A. Frassanito, P. Leone, M. Galiano, V. De Re, F. Silvestris, and F. Dammacco Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules. J. Virol., April 1, 2006; 80(8): 3923 - 3934. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), IgG (
), or IgA (
) determined. The results presented in a and b are from the same experiment. c, CFSE-labeled memory B cells were cultured with CD40L, IL-2, and IL-10 for 5 days. Cells defined according to division history and expression of CD27 and CD38 were sort-purified and recultured for an additional 2 days with CD40L, IL-2, and IL-10. The amount of Ig secreted was determined by Ig H chain specific immunoassays. The values represent the mean Ig production from each population of activated memory B cells from three independent experiments.
, •) represent the percentage of cells migrating in two different experiments; the columns represents the mean of the individual values.