Monocytoid B Cells Are Distinct From Splenic Marginal Zone Cells and Commonly Derive From Unmutated Naive B Cells and Less Frequently From Postgerminal Center B Cells by Polyclonal Transformation

Eleven cases of Piringer’s lymphadenopathy from which paraffin blocks and snap-frozen material were available were taken from the files of the Institute of Pathology at the University Hospital Benjamin Franklin of the Free University of Berlin. All 11 cases were used for immunophenotypical analysis, 6 of them for in situ hybridization and 5 cases with the most prominent areas of monocytoid B cells were selected for the isolation of single cells. For the comparative analysis of mantle cells, germinal center cells, and marginal zone cells, 2 normal spleens and 2 to 4 mesenterial lymph nodes from 2 patients were included in our study.

Immunohistochemical analysis of the monocytoid B cells was performed on formalin-fixed paraffin-embedded tissues. Paraffin sections were pretreated by heating in 0.01 mol/L citrate buffer, pH 6.0, for 2 minutes in a pressure cooker followed by cooling to room temperature and washing with Tris-buffered saline. Frozen sections, immunostained for single-cell isolation, were air-dried overnight and fixed in acetone for 10 minutes. For identification of the monocytoid B cells as well as B-cell subsets and T cells, antibodies directed against the following antigens were applied: CD20 (L26), DBA44, CD25 (Ber-Act 1), CD103 (Ber-Act 8), CD11c (KB90), BCL-2 (124), BCL-6 (594), IgM, IgD, IgA, IgE, CD3 (DAKO, Glostrup, Denmark), CD23 (1B12), CD10 (56C6), CD5 (4C7), CD4 (1F6; Novocastra, Newcastle-on-Tyne, UK), CD27 (1A4CD27; Beckman Coulter Inc, Fullerton, CA), Ki-67 (Mib-1; kindly provided by Prof J. Gerdes, Borstel Institute, Borstel, Germany), and CD45RA (Ki-B332; kindly donated by Prof M.R. Parwaresch, Kiel, Germany). The binding of antibodies was made visible either by the immuno-alkaline phosphatase-antiphosphatase (APAAP) technique or by the biotin-avidin-method, as previously described.33 34 

In situ hybridization for the detection of mRNA specific for IgM, IgD, and IgG was performed as previously described.35 In brief, after linearization of plasmids containing the IgD, M, and G heavy chain constant regions with appropriate restriction enzymes,³⁵S-labeled run-off antisense and sense (control) transcripts were generated using T7 or SP6 RNA polymerases. Paraffin sections were pretreated by exposing them to 0.2 N HCl and 0.125 mg/mL pronase (Boehringer Mannheim, Mannheim, Germany), followed by acetylation with 0.1 triethanolamine, pH 8.0/0.25% (vol/vol) acetic anhydride and dehydration through graded ethanols. Slides were hybridized to 2 to 4 × 10⁵ cpm of labeled probes overnight at 50°C. Washing and autoradiography was performed as described.36 Cells with a 4-fold signal intensity, as compared with background signal, were scored as positive. Hybridization with sense probes only showed homogeneously distributed low background signal. The probes for the Ig heavy chain constant regions were prepared from cDNA of peripheral blood lymphocytes or B-cell lines and cloned into the appropriate plasmids. The nucleotide sequence of the probes was confirmed by DNA sequencing.

Single cells, identified by their localization and their immunophenotype (ie, monocytoid B cells by their negativity for BCL-2 and CD21; mantle cells by their expression of IgD; germinal center cells by their positivity for BCL-6/CD10 and/or Ki-67; and splenic marginal zone cells by their IgM⁺/IgD⁻isotype profile), were isolated from frozen sections, as previously described.37 In brief, immunostained frozen tissue sections were covered with a buffer containing salmon-sperm DNA (1 mg/mL in phosphate-buffered saline [PBS]). Single cells were isolated from the surrounding tissue and transferred into a polymerase chain reaction (PCR) test tube by means of a manipulation capillary and reception capillary, which were fixed to a hydraulic micromanipulator. The whole isolation process was monitored by microscope. Isolated T cells and aliquots of the overlaying buffer drawn after each cell isolation process served as the negative controls.

PCR for the detection of IgH genes was performed as previously described.38-40 In brief, after proteinase K digestion of the isolated cells for 1 hour (1.0 mg/mL), a fully nested PCR using family-specific frame-work (FW) 1 primers for the first amplification and family-specific FW2 primers for reamplifications was used in conjunction with 2 nested primers for the joining region (JH). The first round of amplification was performed with 40 cycles consisting of 5 cycles of 15 seconds at 96°C, 30 seconds at 63°C, and 30 seconds at 72°C, followed by 35 cycles of 15 seconds at 96°C, 30 seconds at 57°C, and 30 seconds at 72°C in 2.5 mmol/L MgCl₂, 200 μmol/L each dNTP, 300 ng of the FW primers (50 ng each), 100 ng of the JH primers, and 2 U Taq DNA polymerase (AmpliTaq; Perkin Elmer, Weiterstadt, Germany). The second round of amplification consisted of 35 cycles of 15 seconds at 96°C, 30 seconds at 63°C, and 30 seconds at 72°C. Six microliters of the reaction mixture was analyzed on an ethidiumbromide-stained polyacrylamide gel (PAGE, 6%). The visualized bands of the PCR products were isolated and directly sequenced in both directions by separate application of the PCR primers. The sequencing was performed with an automated fluorescence DNA sequencer (377A; Applied Biosystems, Weiterstadt, Germany) by using the Dye Deoxy Terminator method.

The amplified VH rearrangements were analyzed for VH usage and for somatic mutations by comparison with germline VH-segments in the VBASE databank, and coding capacity was determined by translation of the sequences into amino acids. To exclude contaminations, all amplified sequences were also compared with our own and published data bank sequences.40 41 

To determine the pattern of somatic mutations compatible with antigen selection, 2 methods were applied. First, the ratio of replacement to silent mutations (R/s) in the CDR2 and FW3 region was studied.42 A sequence was considered to be antigen selected when the R/s ratio in the CDR2 was higher than 2.9 and R/s ratio in the FW3 region was lower than 1.5. Second, only the R/s ratio of the somatic mutations in the FW3 region was considered. A sequence was regarded as being antigen selected when the R/s ratio was less than 1.6.43 

To update the phenotype of the monocytoid B cells and to determine their relationship to other B-cell populations, an immunophenotypical analysis was performed on 11 cases of Piringer’s lymphadenopathy, including new marker molecules (Tables 1 and 2 and Figs 1 and2). This investigation confirmed that monocytoid B cells differ from mantle B cells by a complete absence of BCL-2 (Fig 2b) and CD23 and a vast absence of sIgM and sIgD (Fig 1d and e). They can be distinguished from germinal center B cells by the absence of CD10, BCL-6, cytoplasmic Ig, and partial presence of DBA44, a hairy cell leukemia-associated molecule. From splenic and nodal marginal zone B cells, monocytoid B cells can be discriminated by a partial presence of DBA44, a complete presence of the Ki-B3 epitope of the CD45RA molecule, and additionally by a complete absence of BCL-2 and a nearly complete absence of sIgM. Different from all other B-cell subpopulations, the monocytoid B-cell zone was devoid of T cells and follicular dendritic cells (FDC) (Fig 2a and d), but contained a significant admixture of neutrophils. The proliferation rate of monocytoid B cells, varying from 10% to 25%, was significantly higher than that of mantle B cells (1% to 5%) as well as of splenic and nodal marginal zone B cells (5% to 10%), but considerably lower than that of germinal center B cells (50% to 100%), as demonstrated by the Ki-67 index (Fig 2c).

To check the validity of the Ig labeling results and whether the absence of Ig protein expression on most monocytoid B cells is due to a downregulation at the transcriptional or translational level, the expression of Ig mRNA (Igμ, δ, and γ) was studied and compared with the presence of the corresponding Ig protein (Table 2). Igμ and Igδ mRNA were detectable by in situ hybridization in approximately 20% of the monocytoid B cells, a finding in harmony with that obtained by immunohistochemistry for protein detection (Fig3a). An unexpected result was the finding of an expression of Igγ transcripts on the majority of monocytoid B cells in the absence of IgG protein molecules. The level of this RNA expression was low and corresponded with that of other B cells carrying surface membrane bound Ig (Table 2 and Fig 3b and c).

Three hundred twenty-seven monocytoid B cells were isolated from the lymph node sections of 5 cases of Piringer’s lymphadenopathy. For comparison, single cells were isolated from mantle zones and germinal centers of the same and other cases, as well as from the marginal zone of 2 spleens, and were subjected to a single-copy PCR and sequence analysis for the demonstration of Ig heavy chain gene rearrangements and their mutation pattern. Ninety-five sequences (29%) were obtained from the 327 isolated monocytoid B cells, and 85 of these sequences (89%) were unique. In addition, 5 unrelated sequences occurred twice in 3 of the 5 cases of Piringer’s lymphadenopathy. The sequence comparison of the VH rearrangements with databank germline VH segments showed that 67 (74.4%) of the monocytoid B cells displayed unmutated VH genes ranging from 50% to 86.6% among the 5 cases investigated (Table 3). Twenty-three sequences (25.6%) harbored somatic VH mutations, with an average mutation frequency of 3.8% and ranging from 1 to 14 base substitutions (Table 3).

To check the validity of the results of our VH mutation analysis, 42 mantle zone B cells, 81 germinal center B cells, and 66 splenic marginal zone B cells were isolated and their VH rearrangements were amplified by PCR, resulting in 12, 25, and 21 amplificates, respectively. The sequence analysis of the PCR products showed that none of the VH gene rearrangements amplified from mantle zone B cells contained somatic mutations or a clonal relation. Germinal center B cells displayed mutated VH rearrangements in most instances that were partly clonally related with signs of ongoing mutations as evidenced by their intraclonal diversity. All VH gene rearrangements amplified from the isolated splenic marginal zone B cells were unique. The majority of them harbored somatic VH mutations (Table4).

Our single-cell isolation technique was controlled by the analysis of 36 single T cells and 552 buffer samples that were drawn after each cell isolation. None of these controls gave rise to VH-specific PCR products, indicating that the isolated monocytoid B cells and control B cells were not contaminated by other cells or by their DNA.

Two methods were applied for the estimation of antigen selection in the mutated B cells. Both methods agreed in that the majority of the monocytoid B cells with VH mutations (88.8%; 16/18) lacked signs of antigen selection when the CDR2 and FW3 region were analyzed and 55.5% (10/18) lacked signs when only the FW3 proportion was drawn into consideration. Five sequences with somatic Ig mutations could not be used for the determination of antigen-selection due to their very low number of mutations. In contrast to the monocytoid B cells, most of the mutated splenic marginal zone cells showed signs of antigen selection.

Eighty-three of the 90 individual monocytoid B cells (93%) showed a functional VH rearrangement without stop codons or frame shift mutations. Four nonfunctional VH rearrangements occurred in the CDR3 proportion of the unmutated monocytoid B cells and consisted of 3 stop codons and 2 frame shifts, with 1 cell simultaneously carrying a stop codon in the FW3 region and a frame shift. The remaining nonfunctional VH rearrangement occurred in a mutated monocytoid B-cell and consisted of a deletion and 2 insertions in the FW3 region resulting in a frame shift.

In the present study, we have investigated monocytoid B cells to identify the precursor B cells of monocytoid B cells and to elucidate their relationship to other B-cell populations, including nodal and splenic marginal zone cells. For this purpose, we selected cases of Toxoplasma gondii-induced Piringer’s lymphadenopathy with prominent, and thus reliably identifiable, sheets of monocytoid B cells and analyzed their antigen profile, their Ig isotype expression at the RNA and protein levels, and their Ig gene rearrangement and VH gene mutation pattern. The results obtained were compared with the features of other B-cell populations with special reference to splenic marginal zone B cells and, when technically possible, with the features of nodal cells occurring in the marginal zone of mesenteric lymph nodes. Our immunohistochemical investigations confirmed the vast absence of IgM and the total absence of BCL-2 from monocytoid B cells and the presence of both molecules on marginal zone cells of the spleen and mesenteric lymph nodes. We could further show that the Ki-B3 epitope of the CD45RA molecule and the DBA44 molecule (characteristically associated with hairy cell leukemia cells) were totally absent from splenic and nodal marginal zone cells but were expressed either by all or by a proportion of monocytoid B cells, respectively. The proliferation rate was higher in monocytoid B cells than in splenic and nodal marginal zone cells. Our studies underscore the immunophenotypical differences between monocytoid B cells and all other B-cell subsets (Table 1).

Because the immunohistological demonstration of Ig is prone to artifacts due to high levels of soluble Ig in the serum,44we extended our studies to the expression of Ig mRNA. The in situ hybridization experiments showed, for Igμ and Igδ transcripts, a distribution similar to that of the corresponding proteins, indicating that the vast absence of IgM and IgD in monocytoid B cells is a true finding and that their expression is regulated at the transcriptional level. These findings confirm previous studies showing that monocytoid B cells are distinct from all known B-cell populations, including marginal zone cells of the spleen and mesenteric nodes, and dismiss the concept that monocytoid B cells represent the nodal equivalent of splenic marginal zone cells. An unexpected finding of our in situ hybridization studies was weak, but distinct signals for Igγ over the monocytoid B cells in the absence of IgG protein labeling. However, a similarly weak but distinct labeling for Igγ transcripts was found over most germinal center B cells, with the difference that, in the germinal center light zone, there were some very strong additional Igγ transcript signals. These strong signals could be allocated to germinal center cells being in the process of plasma cellular differentiation. This finding demonstrates the absence of plasma cellular differentiation from monocytoid B cells and thus adds to the differences between monocytoid B cells and germinal center cells. In conclusion, the present and previous phenotypical studies18,19 21 disclosed differences between monocytoid B cells and established B-cell populations and thus proved to be incapable of identifying the B-cell population that gives rise to monocytoid B cells.

We therefore turned our studies to the genetic level. This included the analysis of the rearrangement and mutation pattern of the VH genes in single monocytoid B cells isolated from 5 different cases. Ninety-five amplificates from 327 isolated monocytoid B cells could be obtained. The detection of VH rearrangements in the monocytoid B cells formally proved their B-cell nature. The finding that nearly all VH rearrangements (85/95 [89%]) of the monocytoid B cells were unique indicates that most, if not all, monocytoid B cells originate from many (polyclonal) B cells and not from a few cells or one B cell by oligoclonal or monoclonal expansion. This polyclonal origin of the monocytoid B cells is further substantiated by a VH family usage compatible with normal B cells excluding a derivation from B cells with a particular type of VH rearrangement.

The sequence analysis of the IgH rearrangements showed that most monocytoid B cells (95%) contained functional Ig genes. Of the monocytoid B cells, 74.4% were devoid of VH gene mutation and 25.6% carried somatic mutations at a frequency (3.8%) comparable to those of IgG- and IgA-positive peripheral blood memory B cells.43 45These findings indicate that the monocytoid B cells, despite their uniform morphological appearance, uniform antigen profile, and uniform homing, are not homogeneous but consist of a mixture of B cells, with the majority of them representing B cells at a pregerminal center stage of differentiation, like mantle cells, and a minority representing late B cells that have acquired somatic mutations during their passage through a germinal center, like memory B cells. However, most of the mutated monocytoid B cells lacked signs of antigen selection, drawing their derivation from normally selected memory B cells into doubt.

These findings are in partial conflict with a recent study29 reporting that splenic as well as nodal marginal zone B cells (designated as monocytoid B cells in the mentioned study) show somatic mutations and display clonal expansion. We therefore extended our genetic single-cell studies to splenic marginal zone B cells, mantle cells, and germinal center B cells. Unfortunately, marginal zone cells of mesenteric lymph nodes could not be included because of a lack of frozen tissue. In agreement with previous investigations,43,46,47 mantle cells did not show any clonal relationship or VH gene mutations, but clonally related VH rearrangements with ongoing somatic mutations were detectable in the germinal center cell population. Furthermore, our analysis of the single splenic marginal zone cells showed only unrelated (polyclonal) rearrangements with mostly antigen selected mutations, as shown in a previous study.48 The different results concerning the clonality and mutation pattern of the monocytoid B cells described by Tierens et al29 might have been caused by an erroneous comicrodissection of parts of the germinal centers leading to the appearance of dominant PCR products. This assumption is supported by the reported high frequency of crippling mutations (6 of 16 sequences [37.5%]), because disrupted VH genes to that extent are so far only reported for cells isolated from the inside but not from the outside of germinal centers.43 46 

In conclusion, the absence of related rearrangements from most monocytoid B cells suggests that these cells arise by transformation of many polyclonal B cells, eg, unmutated mantle cells, and to a lesser extent of mutated immature (not fully antigen selected) memory B cells. It is tempting to speculate that this transformation might be induced by a B-cell superantigen expressed by Toxoplasma gondii. It has already been shown that Toxoplasma gondii possess superantigen or superantigen-like activity,49 50 which is effective on T cells and leads to a selective expansion of a certain T-cell subset. It could be that Toxoplasma gondii-expressed superantigens are also active on a subset of pregerminal center and postgerminal center B cells. The postulated superantigen(s) would induce a transformation of B cells of various differentiation stages (ie, unmutated and/or mutated B cells), including a change of their antigen profile and an isotype switch similar to the one seen in germinal centers that is associated with a downregulation of BCL-2, Ig, and CD23 and an upregulation of the proliferation. Furthermore, this superantigen could be responsible for the attraction of neutrophils into the monocytoid B-cell zone.

Taken together, our findings support the view that monocytoid B cells represent a separate B-cell subset that is distinct from all established B-cell subpopulations, including marginal zone cells of the spleen and mesenteric lymph nodes. They are not homogeneous, because 74.4% of monocytoid B cells appear to derive from unmutated naive pregerminal center B cells and 25.6% disclose VH mutations compatible with a derivation from postgerminal center B cells. The predominant expression of Igγ transcripts points towards an uncommon dissociation between isotype switch and somatic VH gene mutation, at least in the unmutated proportion of monocytoid B-cell, further stressing the distinctiveness of these cells.

The authors thank H. Lammert, E. Berg, H. Protz, D. Jahnke, and H.-H. Müller for their excellent technical assistance and L. Udvarhelyi for his help with the preparation of the manuscript.