Published online before print March 12, 2004
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* Deutsches Rheuma-Forschungszentrum, Berlin, Germany;
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany;
Charite’ Humboldt University, Berlin, Germany; and
Orthopädische Klinik Braunschweig, Germany
1 Correspondence: Deutsches Rheuma-Forschungszentrum, Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail: manz{at}drfz.de
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Key Words: antibodies • B lymphocytes • cellular differentiation • memory
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6 months in mice [6
]. Human plasma cell differentiation has been studied extensively in vitro [7 8 9 ] and on plasma cells isolated from blood [10 , 11 ]. The studies on normal plasma cells isolated from tonsils, blood, and bone marrow indicate that these tissues contain plasma cells expressing high levels of CD38 but heterogeneous levels of other surface molecules [12 , 13 ]. Although early plasma cells and their precursors are found in tonsils, more mature plasma cells are present in the bone marrow, possibly reflecting the different functions of these tissues in inducing and maintaining plasma cell responses, respectively. Immunoglobulin G (IgG) is the dominant Ig isotype secreted by these cells in T-dependent immune responses [14 ]. It has been indicated that most IgG-secreting cells present in the tonsils are proliferating cells (B cell/plasma blasts), and IgG-secreting cells in the bone marrow are nonproliferating cells (plasma cells) [12 ]. However, the exact relationship between phenotypically distinct plasma cell populations is not clear yet.
Here, we used the cellular affinity matrix technology to identify human IgG-secreting cells, i.e., plasma cells and their immediate precursors. This technology had been applied initially to identify murine antibody (Ab)-secreting plasma cells and cytokine-secreting T cells [15 ]. We identified subpopulations of IgG-secreting cells, including a CD38 low plasma cell precursor population present in tonsillar germinal centers.
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Cellular affinity matrix technology
Single-cell suspensions were biotinylated with 0.5 mg sulfo–normal human serum–Langerhans cell–biotin (Pierce, Rockford, IL) in 1 ml PBS (37°C, for 10 min), and then the same volume of RPMI 10% fetal calf serum (FCS) was added, and cells were incubated for another 10 min. After three washing steps with PBS/0.5% BSA, cells were incubated with a Cy5-coupled anti-human IgG (PharMingen, BD, San Diego, CA, 10 µg/ml, 15 min on ice). Subsequently, the cells were washed, and unconjugated anti-human 
[clone HP6054 (American Type Culture Collection, Manassas, VA), 50 µg/ml, 0.2 ml, 4°C] was added; 5 min later, 50 µg/ml HP6054 conjugated to avidin [catching Ab (catch-Ab)] was added. After 10 min incubation on ice, cells were resuspended in 2 ml RPMI/10% FCS at a final concentration of 3 million cells/ml and incubated for 30 min at 37°C. Under these conditions, Ig was secreted, and those bearing light chains were captured on the surface of the Ig-secreting cells. Thereafter, the cells were cooled on ice for 10 min, and 5 µg/ml digoxigenized (DIG) anti-human IgG conjugate was added (PharMingen, BD). After washing, cells were stained with a titrated concentration of anti-DIG-phycoerythrine (PE). To allow discrimination among membrane bound and secreted-captured IgG, cells were stained for surface IgG before capturing the secreted IgG.
Monoclonal Ab (mAb) and fluorescein-activated cell sorter (FACS) analysis
The following fluorescein-labeled antibodies were used for phenotyping: CD3, CD5, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD28, CD35, CD40, CD44, CD45, CD49d, CD49e, CD70, CD71, CD72, CD86, CD95, CD100, CD154, and human leukocyte antigen (HLA)-DR (PharMingen, BD); CD9 and CD38 (Immunotech, Coulter, FL); and CD10 and CD138 (AMS Biotechnology GmbH, Germany). A FACScalibur (Becton Dickinson) flow cytometer was used for analysis. Propidium iodide (PI; 1 µg/ml) was used to exclude dead cells according to uptake of the dye. For intracellular staining, cells were fixed in 2% formaldehyde and incubated with anti-IgG-fluorescein [fluorescein isothiocyanate (FITC)] in PBS/BSA containing 0.5% saponin.
In vitro stimulation of PBMC
CD38/CD138-depleted PBMC were labeled with carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) as described [17
] and were subsequently stimulated with 1 µg/ml tetanus toxoid [18
]. To deplete CD38/CD138-positive cells, PBMC were labeled with CD38-PE (PharMingen, BD) and thereafter, with anti-PE and CD138 microbeads. Cells were depleted by magnetic cell sorter (MACS; Miltenyi Biotech). The depleted fraction did not contain any detectable IgG-secreting plasma cells, as tested by the IgG-secretion assay. CD38 high cells were also not detectable by FACS analysis.
Histology
Freshly ectomized human tonsils were cut in convenient pieces of 300 mm3 and embedded in Tissue-Tek® Cryomold® intermediate. Cryosections (7 µm) were performed and acetone-fixed. Before specific Ab staining, unspecific binding sites were blocked with PBS/2% BSA for 20 min at room temperature. Sections were stained with FITC-labeled mouse anti-human CD38 mAb (Dako, Carpinteria, CA; clone AT13/5) and biotinylated mouse anti-human IgG mAb (PharMingen, BD, clone G18-145), respectively. After washing, sections were incubated with rhodamin-labeled streptavidin. Nuclei were counterstained with diamidinophenyl indole (DAPI).
Polymerase chain reaction (PCR)
FACS sorted cells directly into PCR tubes. PCR was performed for ß-actin and Blimp-1 (primers, Blimp-1: 5'-TCGGGTCGTTTACCCCATC-3' and 5'-CACAGCGCTCAGGCCATTA-3'). Reactions were annealed at 55°C and amplified for 35 cycles.
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0.47 ± 0.2%, 0.01 ± 0.1%, and 0.2 ± 0.1% (n
8 for all tissues) among the nucleated cells in tonsils, peripheral blood, and in bone marrow, respectively. Although frequencies in tonsils and bone marrow showed little variation between different donors, frequencies of blood IgG-secreting cells could reach up to 0.1% in a few samples. The expression of various surface markers on IgG-secreting cells was analyzed (Fig. 2
). A substantial fraction of the tonsillar IgG-secreting cells expressed HLA-DR and thus can be considered to be early plasma cells [20
, 21
]. CD38 was expressed at high levels on all IgG-secreting cells derived from blood and bone marrow. However, an additional IgG-secreting cell population was detected in the tonsils, expressing only low levels of CD38. A third population of IgG-secreting cells, present in only very low numbers, expressed even less CD38.
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Figure 1. Detection of IgG-secreting cells. Bone marrow, blood, or tonsil-derived single-cell suspensions (here, cells from bone marrow are shown) were stained for surface and secreted IgG as described in the text. (A) Left dot plot: Cells were stained for surface and subsequently for secreted IgG as described in Materials and Methods. Right dot plot: Negative control treated with all staining reagents but lacking catch-Ab. The IgG-secreting cell population (left plot) is indicated by the square. (B) Cells stained for surface and secreted IgG as shown in A were fixed and stained for intracellular IgG. Saponin was used to allow the staining Ab to penetrate the cell membrane. Nonsecreting/surface IgG+ cells were excluded from the analysis by gating (gate shown in A, left plot). The cells stained for secreted IgG were specifically labeled for intracellular IgG (left) but neither for an isotype control (right) nor for IgG without permeabilization of the cell membrane (middle). Dead cells were excluded from the analysis by PI staining. About 2 x 105 cells are shown in each dot plot. Representative data of 12 independent experiments are shown.
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Figure 2. Identification of IgG-secreting plasma cell subpopulations. Single-cell suspensions from tonsils (left column), blood (middle column), and bone marrow (right column) were stained for surface and secreted IgG and counterstained for the markers indicated. Dead cells were excluded from the analysis by PI staining. Nonsecreting/surface IgG+ cells were excluded from the analysis by gating (shown in Fig. 1A
, left plot). About 2 x 105 cells are shown in each dot plot. Representative data of six independent experiments are shown.
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To further characterize IgG-secreting subpopulations, as a measurement for cell size, we compared the mean forward-scatter profile of these cells with that of B cells (Table 1
). Tonsilar CD38-negative/very low IgG-secreting cells had about the same forward-scatter profile as B cells, i.e., 245 linear units. The CD38 low and CD38 high population showed an increased scatter profile with 302 and 317 linear units, respectively. The differences in scatter among all three populations were statistically significant (P<0.05). These values were still below those measured for IgG-secreting cell populations isolated from blood or bone marrow. As an increase in cell size is probably associated with an increase in plasma cell maturation [23
], these results already suggested that the relatively small, CD38 low Ab-secreting cells were early plasma cell precursors. To further test whether CD38 low IgG-secreting cells resemble a temporary stage of the early plasma cell development rather than a distinct plasma cell subpopulation, we analyzed the expression of this marker on stimulation of B cells in vitro. PBMC were depleted of plasma cells and activated B cells by MACS using CD38 and CD138 magnetic beads. The remaining cells, consisting mainly of resting B cells (memory and naive), were activated by tetanus toxoid as described [18
]. When IgG-secreting cells first appeared at day 3 in culture, these cells expressed no or only low levels of CD38 (Fig. 3A
). Later, at day 6, all IgG-secreting cells had up-regulated CD38 to high levels. During the same period of time, the frequencies of these cells increased from 0.2 to 1.8%. The staining for secreted IgG on individual cells also increased between days 3 and 6 in culture, suggesting an increase in IgG secretion and level of maturation. Labeling the cells with CFSE allowed the analysis of their proliferation profile [17
]. Already at day 3, the early-appearing IgG-secreting cells had lost some CFSE, indicating that proliferation precedes differentiation into IgG-secreting cells (Fig. 3B)
. Until day 6, the IgG-secreting cells underwent further rounds of proliferation, indicated by a further decrease in CFSE labeling. These data support the idea that plasma cell differentiation starts already at a CD38-negative stage.
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Table 1. Mean Forward Light-Scatter Analysisa
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Figure 3. Plasma cell differentiation in vitro. (A) PBMC were depleted of plasma cells and stimulated with tetanus toxoid. IgG-secreting cells were identified by the cellular affinity matrix technology as described in the text and Figure 1
. At the time-points indicated, the IgG-secreting cells were stained for CD38. (B) PBMC were labeled with CFSE, and cultures were initiated as described in A. Cells were analyzed at the indicated time-points. Dead cells were excluded from the analysis by PI staining. Representative data of three independent experiments are shown.
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Figure 4. Phenotype of CD38-intermediate CD9/CD19-positive tonsilar B cells. (A) Tonsillar cells were stained for CD9, CD19, and CD38 together with CD27 or CD138. CD19-negative cells were excluded from the analysis. (B) FACS sorted tonsilar cell populations, and PCR was performed on the indicated number of cells. Lanes 4/5 and 6/7 represent probes obtained from two different donors analyzed in one experiment. Data shown are representative of four independent experiments.
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Figure 5. CD38 and IgG staining on tonsillar sections. Cryosections were stained with mouse anti-human mAb for CD38 (FITC, green) and IgG (rhodamin, red); nuclei were stained with DAPI (blue). Cells expressing high levels of CD38 together with IgG appear in yellow.
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Colocalization of interstitial IgG and CD-38 low IgG-containing cells indicates that these cells already had started to secrete IgG and thus were committed to plasma cell differentiation. This result is in accord with the observation that Blimp-1, a master factor for plasma cell differentiation, is expressed in a fraction of 4–15% of germinal center B cells [26 ].
We further characterized the phenotype of blood IgG-secreting cells in donors not recently vaccinated. In contrast to the observed heterogeneity within the tonsilar and bone marrow IgG-secreting cell compartment, IgG-secreting cells in the blood were homogenous with respect to the expression of most markers analyzed (Table 2 ). The phenotype of steady-state peripheral blood IgG-secreting cells observed here does not differ from that described for blood plasma cells induced by immunization or that described for peripheral blood plasma cells in systemic lupus erythematosus patients [10 , 13 , 27 ].
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Table 2. Phenotype of Peripheral Blood Ig-Secreting Cellsa
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A fraction of the plasma cells formed in secondary lymphoid tissues can enter the bloodstream to migrate to the bone marrow, an organ, which is of particular importance for the survival of long-lived plasma cells. That bone marrow plasma cells are the progeny of B cells activated in secondary lymphoid tissues has been known [3 ]. Also, it has been shown that Ab-secreting cells present in secondary lymphoid tissues following immunization are equipped with chemokine receptors, allowing their emigration into the bone marrow, intestinal lamina propria, or into inflamed tissues [1 , 2 , 30 ], suggesting that the formation of Ab-secreting cells and their early differentiation occurs in secondary lymphoid tissue. It is interesting to note that peripheral IgG-secreting cells in the blood showed a homogeneous phenotype, and their progeny and their precursors, i.e., bone marrow plasma cells and tonsillar plasma cells, are heterogeneous with respect to CD9, CD19, CD38, and CD45. This suggests that plasma cell interaction with stromal cells, extracellular matrix components, and/or cytokines present in lymphoid tissues may regulate the expression of various surface molecules on plasma cells. That plasma cells are responsive to extrinsic signals had been shown earlier. Tonsillar plasma cells, e.g., can be rescued from apoptosis by stromal cells [31 ]. Also, the interaction with their environment allows the survival and proliferation of plasma blasts [32 ] and the entrance of a certain number of these cells into the long-lived plasma cell compartment in the splenic red pulp [33 ].
As a result of the absence of CD19 on myeloma cells, plasma cell differentiation has been considered to be accompanied by the loss of this molecule [34 , 35 ]. In accord with other reports [13 , 27 ], we show that CD19 is expressed on all nonmalignant, IgG-secreting cells including the later plasma cell stages in the bone marrow. However, the level of CD19 expression on IgG-secreting cells differs in the analyzed tissues. All tonsillar IgG-secreting cells, at least partially resembling early plasma cell stages, express CD19 at high levels, comparable or higher than other CD19-positive cells in this tissue. IgG-secreting cells in the blood, possibly on their way from secondary lymphoid tissues to the bone marrow and other peripheral tissues, show reduced levels of CD19. In the bone marrow, however, two populations of plasma cells are found expressing high or low levels of CD19. The later seem to express CD19 in levels comparable with those expressed on plasma cells isolated from blood. As plasma cells migrating through the blood are at least partly precursors of bone marrow plasma cells, this may indicate that CD19 is lost transiently during plasma cell translocation through the blood but is up-regulated on mature bone marrow plasma cells. This hypothesis is in accord with the observation that the level of CD19 expression on bone marrow plasma cells correlates with their cell size (Table 1) and thus with an increase in the level of maturation [23 ]. However, this issue remains to be further elucidated.
Our data show that plasma cell differentiation can be dissected in distinct, developmental stages of CD38 low and CD38 high cells. The presence of distinct, sequential steps during early plasma cell differentiation in the tonsils may possibly mark controlled checkpoints regulating this process in secondary lymphoid tissues. The presence of distinct subpopulations of bone marrow IgG-secreting cells representing later stages of the plasma cell development may indicate the presence of distinct types of plasma cells, e.g., short-lived and long-lived ones. However, we did not find subpopulations among the bone marrow plasma cell precursors, i.e., the IgG-secreting cells in the peripheral blood, arguing against different plasma cell lineages. An alternative explanation for the presence of multiple subpopulations of bone marrow plasma cells is the presence of a "checkpoint" controlling the entrance into the long-lived plasma cell compartment. Such a mechanism would explain the distinct bone marrow plasma cells by the presence of a population, which has entered the bone marrow just recently but is not allowed to enter the long-lived compartment yet, and a plasma cell population resembling the long-lived compartment. This idea is in accord with the hypothesis that plasma cell longevity is not guaranteed by entering the bone marrow but depends on specific niches providing survival signals [36 37 38 39 ].
Received June 18, 2003; accepted January 21, 2004.
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