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The Journal of Immunology, 2007, 179, 5326 -5334
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Population of CD19highCD45R–/lowCD21low B Lymphocytes Poised for Spontaneous Secretion of IgG and IgA Antibodies1

Belén de Andrés2,*, Isabel Cortegano{dagger}, Natalia Serrano*, Borja del Rio*, Paloma Martín{ddagger}, Pilar Gonzalo3,*, Miguel A. R. Marcos{dagger} and María Luisa Gaspar2,*

* Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; {dagger} Centro de Biología Molecular S.O., Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain; and {ddagger} Hospital Puerta de Hierro, Instituto Madrileño de la Salud-Universidad Autónoma de Madrid, C/San Martín de Porres, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ab responses to selected Ags are produced by discrete B cell populations whose presence and functional relevance vary along the ontogeny. The earliest B lineage-restricted precursors in gestational day 11 mouse embryos display the CD19+CD45R/B220– phenotype. Phenotypically identical cells persist throughout gestation and in postnatal life, in parallel to the later-arising, CD19+CD45R+ B cells. Very early after birth, the CD19+CD45R– B cell subset included high frequencies of spontaneously Ig-secreting cells. In the adult spleen, a small subset of CD19highCD45R–/lowIgM+/–IgD–CD21/Cr2–/low cells, which was detected in perifollicular areas, displayed genetic and phenotypical traits of highly differentiated B cells, and was enriched in IgG- and IgA-secreting plasma cells. In vitro differentiation and in vivo adoptive transfer experiments of multipotent hemopoietic progenitors revealed that these CD19highCD45R–/low B cells were preferentially regenerated by embryo-, but not by adult bone marrow-, derived progenitors, except when the latter were inoculated into newborn mice. Both the early ontogenical emergence and the natural production of serum Igs, are shared features of this CD19highCD45R–/low B cell population with innate-like B lymphocytes such as B1 and marginal zone B cells, and suggest that the new population might be related to that category.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Most Ab responses in the adult are produced by the predominant subset of CD21intCD23+ follicular (FO)4 B lymphocytes during the germinal center (GC) reaction, where T cell help is required, and the processes of Ig class switching, somatic hypermutation, and affinity maturation take place (1). The splenic marginal zone (MZ), which is rich in afferent vessels and surrounds the lymphoid follicle, contains a subset of nonrecirculating CD21highCD23– B cells, prepared for the rapid control of blood-borne, opsonized pathogens (2, 3). Finally, the population of B1 cells is concentrated in the adult coelomic cavities (peritoneal and pleural exudates) and is responsible for the production of most natural serum IgM and of Ab responses to early scavenged pathogens (4, 5). Whereas FO B cells show extensive Ig clonotype diversity and are involved in T cell-dependent, high-affinity Ab responses, both B1 and MZ B cells are considered to represent innate-like lymphocytes, enriched in evolutionarily selected Ig specificities, and are prepared for responses to pathogen-associated patterns. Interestingly, these latter B cells are preferentially generated early in life and may display a potential for self-renewal by which they maintain their population sizes along the life span (6, 7).

The development of lymphoid programs begins in midgestation mouse embryos, when oligolineage lymphoid precursors are detected in fetal liver (FL) and extraliver locations (8, 9, 10). The earliest B cell-restricted, Pax-5-dependent, cKit+AA4.1+CD19+ precursors found in gestational day (E) 11 mouse embryos characteristically lacked the CD45R/B220 pan-BCR (11). CD19+CD45R+ B-lineage precursors did not appear up to E13-E14, and rapidly dominated the B cell compartments of FL and adult bone marrow (BM). However, a subset of CD19+CD45R– cells was maintained and rapidly matured along the gestational and postnatal life periods, both in BM (12) and in spleen. Whereas it has been shown that the adult BM CD19+CD45R– cells include precursors for the B1b cell population, we report here a phenotypically similar population of extrafollicular splenic B cells (CD19highCD45R–/lowIgM+/–IgD–CD21/Cr2–/low), which was greatly enriched in plasma cells spontaneously secreting IgG and IgA isotypes, in the absence of any intentional immunization. In vitro short-term differentiation and in vivo adoptive cell transfers of multipotential hemopoietic progenitors (c-Kit+Sca-1+lineage– (KSL)) revealed that the adult CD19highCD45R–/low splenic B cells preferentially derived from hemopoietic multipotential progenitors of the early mouse ontogeny.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cell suspensions, and in vivo adoptive transfers

BALB/c, C57BL/6 (B6, CD45.2), B6.CD45.1, B6.GFP (13), RAG-2/{gamma} chain–/– (14), CD19–/– (15), CBA/CaJ and CBA/CaHN mice were maintained in the animal facilities of the Instituto de Salud Carlos III (ISCIII) and the Centro de Biología Molecular S.O. (CBMSO). The gestational age was determined by the observation of the vaginal plug after overnight mating (E0). Embryo and adult cells were obtained as described (16, 17). In vivo reconstitution experiments were done by i.v. inoculation of 5 x 103 purified KSL progenitors from congenic donors together with 106 total BM cells of the host genetic background (B6), into either adult B6 irradiated hosts (8.5 Gy, from a 137Ce source, IBL 437C; CIS Biointernational), or adult RAG-2/{gamma} chain–/– and newborn B6-irradiated hosts (2.5 Gy). All animal studies were approved by the ISCIII and CBMSO Ethics Committees and performed in accordance to the Spanish animal protection law.

Flow cytometry analyses and cell purifications

mAbs to CD4 (GK1.5), CD9 (KMC8), CD11b/Mac-1 (M1/70), CD19 (1D3), CD21/Cr2 (7G6), CD23 (B3B4), CD44 (IM7), CD45R/B220 (RA3-6B2), CD117/c-Kit (2B8), CD127/IL-7R{alpha} (B12-1), CD138/syndecan-1 (281.2), IgDa (AMS 9.1), Gr-1 (RB6-8C5), Ter-119 (TER-119), and isotype control mAbs (RB5-95, RG7/1.30, A95-1, A119-1, and G155-178), labeled with FITC, biotin, PE, or allophycocyanin, were purchased from BD Pharmingen. PE-, FITC- and PerCP-conjugated streptavidin were obtained from Southern Biotechnology Associates and BD Pharmingen. mAbs to CD24 (J11d), CD43 (S7), IgM (331.12), CD45.1 (A20), and Sca-1 (D7) were purified from hybridoma supernatants and labeled with FITC or biotin. Nonspecific background was eliminated by incubation with 10% mouse serum and Fc-block (BD Pharmingen). Cell debris and dead cells were discarded by light parameters and by propidium iodide staining. Labeled cells were analyzed in a FACSCalibur (BD Biosciences), with the CellQuest (BD Biosciences) software. Lineage-negative (Lin–) cells were obtained by negative selection after staining with biotinylated, lineage-specific mAbs (Ter-119, CD19, Gr-1, CD4, Mac-1, and CD45R), followed by streptavidin-conjugated magnetic beads (Dynal Biotech). The Lin– cells were then stained with mAbs to c-Kit and Sca-1 Ags, and K+S+L– multipotential progenitors were purified in a DIVA cell sorter (BD Biosciences), under sterile conditions. Splenic CD19highCD45R–/low, CD19+CD45R+, CD21high CD23– (MZ), and CD21intCD23+ (FO) B cells were identified with CD21-FITC, CD19-PE, CD45R-allophycocyanin, and CD23-biotin mAbs followed with streptavidin-PerCP, and were purified as above. The degree of purity from recovered cells was controlled in the FACSCalibur, and only samples over 98% pure were considered valid.

Spontaneous proliferation assay

Triplicate cultures of purified B cell subsets (4 x 104/well) were performed and pulsed with [3H]thymidine (1 µCi/well; Amersham Biosciences) for 12 h. Cells were harvested and [3H]thymidine uptake was measured in a Microbeta 1450 Trilux counter (PerkinElmer).

RT-PCR and real-time PCR

Total RNA was extracted, oligo dT cDNA samples were prepared with AMV-RT, and standard PCR amplifications were performed, as described (11). For each PCR, 10% of the cDNA was amplified by using AmpliTaq Gold DNA polymerase (Roche Molecular Systems) in a PTC-200 DNA Engine cycler (Bio-Rad) for 35 cycles (hypoxanthine guanine phosphoribosyl transferase (HPRT)) or for 40 cycles (Pax-5, Blimp-1, XBP-1, VH7183, and VHJ558). The annealing temperature was 60°C for HPRT, Pax-5, Blimp-1, and XBP-1 amplifications, and 65°C for VH7183 and VHJ558 amplifications. The specific primer pairs used were: HPRT sense, 5'-TGTCATGAAGGAGATGGGAG-3' and antisense, 5'-GCCTGTATCCAACACTTCGA-3'; XBP-1 sense, 5'-TGCTGAGTCCGCAGCACTCAG-3' and antisense, 5'-CGAAGTGGTCCACACGTGGAG-3'; and Cµ antisense, 5'-GGGAAATGGTGCTGGGCACCAA-3'. The primers for Pax-5, Blimp-1, and sense VH7183 and VHJ558 were described (18, 19). The amplification products were separated on 2% agarose gels. Quantitative real-time PCR was performed to detect VpreB, {lambda}5, and HPRT transcripts using the Lightcycler 2.0 detection system (Roche) and the Lightcycler FastStart DNA Master SYBR Green I kit. In this case, the cycling parameters were: 1 cycle of 95°C for 10 min, followed by 45 cycles of 95°C for 20 s, 60°C (HPRT), 63°C (VpreB) and 65°C ({lambda}5) for 30 s, 72°C for 30 s, and 1 cycle of 72°C for 14 min. The primers for VpreB and {lambda}5 were described (11). The relative amount of specific cDNA on each sample was determined on the basis of the specific signal for VpreB or {lambda}5, relative to that of HPRT, using the Lightcycler Relative Quantification software (version 1.01).

In vitro cell differentiation assays

Purified KSL progenitors from E11, E15 FL, and adult BM were seeded on ST-2 stromal cell-coated 24-well plates at 104 cells/ml and supplemented with 3 ng/ml IL-7. Flow cytometry analyses were performed at 3 and 6 days of culture. LPS blasts were obtained by culturing total splenocytes (106 cells/ml) for 72 h, as described (18).

Immunofluorescence and confocal microscopy

Immunofluorescent staining was performed using 4-µm frozen spleen sections that were air dehydrated and fixed in 4% paraformaldehyde. Sections were submitted to 50 mM NH4Cl, and blocked with 10% mouse serum and with the Biotin Blocking System (DakoCytomation). Two-color CD19/CD45R analyses were done by using the tyramide signal-amplification system (PerkinElmer). Endogenous peroxidase activity was quenched with hydrogen peroxidase. The slides were labeled with biotinylated anti-CD19 mAb, revealed with HRP-conjugated streptavidin followed by Cy3-tyramide, and with Cy5-labeled anti-CD45R mAb. For three-color CD19/CD45R/MOMA-1 analyses, the samples were incubated as follows: 1) FITC-labeled anti-CD19 mAb (6D5; Southern Biotechnology Associates), revealed with goat anti-FITC Alexa 488-labeled Ab (Invitrogen Life Technologies); 2) biotinylated anti-MOMA-1 mAb (Acris Antibodies) revealed with streptavidin-conjugated Alexa 546 (Invitrogen Life Technologies); and 3) Cy5-labeled anti-CD45R mAb. Isotype-matched nonspecific mAbs were used as single-color controls or in combination with the other specific mAbs. The slides were mounted in FluorSave Reagent (Calbiochem). The images were acquired with the Laser Sharp 2000 software (Bio-Rad) in a Radiance 2000 AGR-3 confocal (Bio-Rad) attached to an ECLIPSE TE 300 microscope (Nikon), using x40 oil immersion lenses, and merged using the Adobe Photoshop 7.0 software (Adobe Systems). The background brightness intensity of the green channel was fixed by the FITC-CD19 levels obtained in spleen sections from CD19–/– mice. The numbers of CD19highCD45R–/low cells were counted by two observers. Cytospins from sorted cells (Shandon Cytospin 4; Thermo Electron) were stained with FITC-labeled goat anti-mouse IgM and with rhodamine-labeled goat anti-mouse IgG (Southern Biotechnology Associates), examined under a Leitz DMRD microscope (Leica), and the images were captured with the IM500 software (Leica). The Ki-67 nuclear Ag was detected on cytospin preparations after hydrolysis by microwave heating, blockade in 1% BSA, and incubation with anti-Ki-67 mAb (BD Pharmingen) followed by Alexa 488-labeled anti-IgG1 Ab (Invitrogen Life Technologies).

ELISPOT assays

Ig isotype-specific secreting cells were quantified on 96-well plates coated with 10 µg/ml of either purified rat anti-mouse IgM, goat anti-mouse IgG3, or goat anti-mouse total Ig (Southern Biotechnology Associates) overnight at 4°C. After blockade with 1% gelatin/PBS, serial dilutions from purified cell populations were cultured in triplicates overnight at 37°C. Cells were incubated with biotinylated rat anti-mouse {kappa}-chain mAb (187.1) for IgM detection, and with goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgA (Southern Biotechnology Associates), for detection of the corresponding Ig isotypes, or with a mixture of the anti-IgG isotype Abs for detection of total IgG-secreting cells, and then revealed with streptavidin-conjugated alkaline phosphatase (1 h, 37°C; Southern Biotechnology Associates) and 5-bromo-4-chloro-3-indolyl-phosphate in 4% low-melt agarose (Sigma-Aldrich).

Statistical methods

The numbers shown throughout the text represent means ± SD that were obtained with Prism 3.0 (GraphPad Software). The two-tailed Student t tests were used to calculate p values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Two independent CD19+CD45R– and CD19+CD45R+ B-lineage cell populations in FL and adult BM

It was known that CD45R/B220 Ag levels progressively increase in B-lineage cells during late fetal ontogeny (20). We previously described that the sole B-lineage cells detected in E11–13 mouse embryos displayed the CD19+CD45R– phenotype (11). CD19+CD45R+ cells were absent up to E14, when they appeared and rapidly expanded to become the major B cell subset in late gestation and in adult BM. However, CD19+ cells bearing surface levels of CD45R that ranged from negative in earlier (E11–14 FL) to low in later samples (>E16 FL, adult BM) always persisted (Fig. 1A). The number of CD19+CD45R– cells reached a plateau in the E15 liver (97000 ± 7500 cells/liver; n = 10 liver cell pools) and were also present in adult BM, as described (21). The sequential appearance of the CD19+CD45R– and CD19+CD45R+ cell subsets might suggest that the latter population differentiated from the former one. To investigate this possibility, we studied the maturation stage of both cell subsets in E14 FL. The differential expression of CD43, CD25, CD138, and MHC class II surface Ags distinguishes B-lineage differentiation stages in adult BM, but most of these markers were absent from fetal B cell precursors (22, 23) (and our unpublished data). CD117/c-Kit and CD127/IL-7R{alpha} receptors are specific to multipotent and lymphoid-restricted progenitors, respectively, and were differentially expressed by the two subsets of E14 B-lineage cells. Whereas E14 CD19+CD45R+ cells were CD117+CD127+, most of the simultaneous CD19+CD45R– cells had down-regulated expression of both receptors (Fig. 1B). At E11, CD19+CD45R– cells were, in contrast, uniformly CD117+CD127+ (11). The relative transcript levels of the pre-BCR-encoding {lambda}5 and VpreB genes were also analyzed in both FL B cell subsets by quantitative RT-PCR. {lambda}5 and VpreB transcripts were present in purified E13 CD19+CD45R– and markedly increased at E14 in the same population. By contrast, the E14 CD19+CD45R+ cell subset showed much lower {lambda}5 and VpreB transcript levels than those of the simultaneous CD19+CD45R– cell subset. At E18, the expression of both transcripts was equivalent in the two populations (Fig. 1C). Finally, the expression of representative IgH VDJ gene rearrangements (VH7183, VHJ558) was also analyzed in the E14 CD19+CD45R– and CD19+CD45R+ cell subsets: both IgH VDJ transcripts, and especially those of the VH7183 family, were found in the E14 CD19+CD45R– but not in the CD19+CD45R+ B-lineage cells; the two cell types displayed IgH VDJ transcripts 2 days later (Fig. 1D and B. de Andrés, M. A. R. Marcos and M. L. Gaspar, unpublished results). Taken together, the down-regulation of CD117 and CD127 receptors, the higher transcript levels of pre-BCR-encoding genes and the selective transcription of IgH VDJ genes in E14 CD19+CD45R– cells, but not in the simultaneous CD19+CD45R+ cells, support the notion that the former represents a more mature B-lineage cell population at that time point. It is thus unlikely that the E14 CD19+CD45R+ cells differentiated from the earlier-arising CD19+CD45R– cell population. Clonal assays in stromal cell cultures supplemented with IL-7 further revealed the B cell precursor character of both E14 FL B-lineage cells, with the CD19+CD45R– cell subset generating 2- to 3-fold B cell clonal frequencies compared with those of CD19+CD45R+ cells (data not shown).


Figure 1
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  		FIGURE 1. Ontogeny of CD19+CD45R– and CD19+CD45R+ B-lineage cell populations. A, CD19/CD45R stainings in FL and adult BM cells. The window defines the CD19+CD45R– cell population. Numbers are mean ± SD of CD19+CD45R– cells; n = 4 samples/point. B, Three-color FACS analyses of CD117 and CD127 Ag expression in both CD19+CD45R– and CD19+CD45R+ cell subsets of E14 FL. Empty and shaded histograms are irrelevant, isotype-matched, controls and specific Abs, respectively. C, Relative transcript levels of VpreB, {lambda}5, and HPRT genes obtained by real-time RT-PCR in E13, E14, and E18, purified CD19+CD45R– ({square}) and CD19+CD45R+ (Figure 1) FL cells; a.u., arbitrary units. Data are from one representative experiment of three. D, VH7183, VHJ558, VDJ, and HPRT gene transcripts in purified E14 B-lineage cell subsets and in E19 total FL.

 
A population of CD19highCD45R–/lowCD21–/low B lymphocytes in the adult spleen

Two-color CD19/CD45R flow cytometry analyses of normal adult spleen revealed the presence of three additional cell populations, besides the major CD19+CD45R+ B cells and the double-negative, CD19–CD45R– (mostly T) cells: CD19highCD45R–/low, CD19lowCD45R–/low, and CD19–CD45R+ cells (Fig. 2, dot plot). The latter cell subset mostly corresponded to NK cells, as demonstrated by their expression of the NK1.1 Ag in B6 mice (data not shown). The small CD19highCD45R–/low cell subset (0.52 ± 0.11% of total splenocytes; n = 8) displayed several phenotypic differences when compared with the CD19+CD45R+ cells (Fig. 2, histograms): all splenic CD19+CD45R+ B cells were IgM+IgD+, but only 40 ± 15% of the CD19highCD45R–/low cells expressed surface IgM and none displayed IgD. Whereas spleen CD19+CD45R+ B cells were distributed between CD21intCD23+ FO and CD21highCD23– MZ B cell subsets, CD19highCD45R–/low cells were CD21–/lowCD23–/low. The hyaluronic acid receptor (CD44) is up-regulated in activated B cells (24), and was highly expressed in CD19highCD45R–/low compared with CD19+CD45R+ B cells. Furthermore, the splenic CD19highCD45R–/low B cells showed reduced levels of CD24 (HSA), expressed CD43 (leukosyalin) and the CD9 tetraspanin, and contained fractions of CD138 (syndecan-1)+ cells, all criteria compatible with highly differentiated B cells, and that were absent from CD19+CD45R+ mature B cells. They also had reduced levels of class II MHC Ags and did not express CD5 and CD11b (Mac-1) Ags, present in B1 cells (data not shown). In contrast to phenotypically similar BM cells (21) or to those of embryo liver, the splenic CD19highCD45R–/low B cells did not exhibit any B cell progenitor character. They represented a population of high forward scatter (FSC), blastic cells (Fig. 3A). As defined by both the frequencies of cycling, Ki-67+ cells, and by their spontaneous proliferation rates, CD19highCD45R–/low B cells were an actively proliferating cell subset, when compared with splenic FO B cells (n = 3, p < 0.001, Fig. 3B). Preliminary pulse-chase BrdU analyses further suggested that they display high cell turnover rates (data not shown).


Figure 2
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  		FIGURE 2. Cell surface phenotype of CD19highCD45R–/low, CD19+CD45R+, CD19lowCD45R–/low, and CD19–CD45R+ cell populations in the spleen of adult, unimmunized mice. The results were obtained by three-color FACS analyses, and the histograms show the Ag expressions inside the electronic windows defined in the upper dot plot. Shaded and empty histograms are as in Fig. 1. Data are from one representative experiment.

 

Figure 3
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  		FIGURE 3. Cell proliferation state of splenic FO, MZ, and CD19highCD45R–/low B cells. A, FSC values of the purified spleen cells. The dotted line represents the peak fluorescence intensity of FO B cells. B, Left columns, Frequencies of Ki-67+ cells in cytospin samples of purified B cell subsets (6 x 102–103 cells/sample were counted; mean ± SD, n = 3). Right columns, Spontaneous proliferation rates of the purified B cell subsets after a 12-h pulse of [3H]thymidine. The differences in the results obtained with FO vs those of MZ and CD19highCD45R–/low B cells were statistically significant (p < 0.001). Data are representative of three independent experiments and indicate the mean ± SD of triplicate results in each experiment.

 
The CD19lowCD45R–/low cell population, which presumably includes B cells in the transition to the plasma cell stage (25), was easily singularized from the CD19highCD45R–/low cell subset and, although they both share several phenotypical traits, CD19lowCD45R–/low cells had lower levels of surface IgM and CD9, and they included a population of IgD+ cells (Fig. 2).

The CD19highCD45R–/low B cell subset was also found in early postnatal periods. As in adult mice, only a fraction of the postnatal CD19highCD45R–/low B cells became surface IgM+. IgD, however, was transiently expressed in 20–30% of them in 40-day-old mice, to be down-regulated afterward. The CD21/Cr2 receptor was always either absent or expressed at very low levels (Fig. 4).


Figure 4
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  		FIGURE 4. Ontogeny of CD21, IgM, and IgD expressions in CD19highCD45R–/low and CD19+CD45R+ splenic B cells from 15- to 50-day-old BALB/c mice. The data shown are from a representative experiment of three to five performed with spleens from mice at each age. The upper and bottom histograms of each sample correspond to the specific labelings of both CD19highCD45R–/low and CD19+CD45R+ B cells, respectively, as defined in the electronic windows of the left dot plots. Shaded and empty histograms are as in Fig. 1.

 
CD19highCD45R–/low B cells selectively homed to splenic perifollicular areas

The adult spleen is divided into red- and white-pulp areas. The latter is organized in lymphoid follicles with a central B cell area, encircled by the MZ, where afferent lymphatics and T cell-rich periarteriolar lymphoid sheaths are located (26). We investigated whether the splenic population of CD19highCD45R–/low cells was preferentially found in a particular spleen location, by means of CD19, CD45R, and MOMA-1 stainings, and confocal microscopy. To detect the dull signals of anti-CD19 mAbs, we revealed them with either a tyramide signal-amplification system or with the use of a secondary anti-FITC mAb. Spleen sections from CD19-deficient mice (15) submitted to the CD19-staining protocol, as well as the isotype-matched controls, were negative (data not shown). The vast majority of splenic B cells coexpressed CD19 and CD45R surface receptors, as expected (yellow cells; Fig. 5, A and B). CD19highCD45R–/low B cells were found particularly concentrated in the periphery of spleen follicles, at both sides of the MOMA-1+ band, which defines the internal border of the MZ (9.2 ± 3.15 cells/field of 50 x 50 µm; Fig. 5, A and B, arrows in the enlarged boxes), less frequently in the central follicular areas (3.1 ± 0.6 cells/field) (n = 43 fields, in eight follicles) and rarely in the red pulp (Fig. 5C).


Figure 5
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  		FIGURE 5. In situ localization of splenic CD19highCD45R–/low B cells. A, Two-color analyses of biotinylated anti-CD19 revealed with the tyramide signal-amplification system (green), and Cy5-labeled anti-CD45R (red) mAbs. B, Three-color stainings of biotinylated MOMA-1 revealed with streptavidin-Alexa 546 (blue), FITC-labeled CD19 (green), Cy5-labeled CD45R (red) mAbs. The areas defined in the boxes are enlarged in the right panels, showing the isolated CD19 and CD45R signals, and merged views. C, CD19/CD45R signals observed in extrafollicular areas of the spleen. White arrows point to representative CD19highCD45R–/low B cells. Scale bars, 40 µm.

 
Splenic CD19highCD45R–/low cells were enriched in natural plasma cells spontaneously secreting IgG and IgA Abs

To establish the differentiation stage and functional activity of splenic CD19highCD45R–/low B cells, we purified them along with FO (CD21intCD23+) and MZ (CD21highCD23–) B, CD19–CD45R– splenic B cells, and LPS blasts as controls, and measured the transcription levels of Pax-5, Blimp-1, and XBP-1 genes by RT-PCR (Fig. 6A). FO and MZ B cells clearly expressed Pax-5, but the late B cell-specific Blimp-1 and XBP-1 gene transcripts were undetectable in FO and slightly positive in MZ B cells. By contrast, CD19highCD45R–/low splenocytes expressed lower Pax-5, and high Blimp-1 and XBP-1 transcript levels, a typical pattern of late B cells in their way to Ig-secreting plasma cells. Actually, IgM- and IgG-secreting plasma cells were observed by immunofluorescence microscopy in cytospin preparations of purified CD19highCD45R–/low splenic cells, as cytoplasmic IgM+ and IgG+ large cells, respectively, whereas CD19+CD45R+ cells predominantly showed a weak membrane IgM signal (Fig. 6B). We then analyzed the spontaneous Ig secretion rates of purified CD19highCD45R–/lowIgM+, CD19highCD45R–/lowIgM–, and of total spleen cells by means of Ig isotype-specific ELISPOT assays (Fig. 6C). IgM-secreting cells were modestly enriched among CD19highCD45R–/lowIgM+ cells (4.5-fold the total spleen frequencies), but undetectable in CD19highCD45R–/lowIgM– cells (<1 in 105 seeded cells). By contrast, high frequencies of IgG- and IgA-secreting cells were present in the CD19highCD45R–/low cell population, in particular, in the CD19highCD45R–/lowIgM– cell subset. Purified CD19highCD45R–/lowIgM+ spleen cells were moderately enriched in IgG1- and IgA-secreting cells when compared with total spleen cells (2.5- and 1.7-fold, respectively) and more in IgG2a and IgG2b Ab-secreting cells (10.4- and 21-fold, respectively), and they did not produce IgG3. Purified CD19highCD45R–/lowIgM– cells were much more enriched in IgG- and IgA-secreting cells, in a range going from 12- and 33-fold increases of total spleen frequencies for IgG1 and IgG3, respectively, up to a 56-fold increase for IgA-secreting cells. In terms of their contribution to the absolute numbers of splenic plasma cells, the minor CD19highCD45R–/low B cell population accounted for only 2.1 ± 0.3% of all IgM-secreting cells, but they included significant fractions of total spleen IgG- and IgA-secreting cells, ranging from 11.2 ± 3% for IgG1 to 24.2 ± 2.4% and 26.1 ± 6.2% for IgG2a and IgA, respectively. In contrast, CD19+CD45R+ splenic B cells did include very low frequencies of plasma cells (63 ± 9, 42 ± 3, and 59 ± 8 x 10–5 cells for IgM, total IgG, and IgA, respectively), which were concentrated among T cell-depleted CD19–CD45R– splenic cells (data not shown).


Figure 6
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  		FIGURE 6. Differentiation stage and spontaneous Ig secretion of spleen CD19highCD45R–/low cells. A, Transcript levels of Pax-5, Blimp-1, XBP-1, and HPRT genes in purified CD19highCD45R–/low, FO (CD19+CD45R+CD21intCD23+), and MZ (CD19+CD45R+CD21highCD23–) B cells, CD19–CD45R– splenocytes and 72-h LPS blasts of adult BALB/c mice. B, Cytospin preparations of purified CD19highCD45R–/low and CD19+CD45R+ spleen cells, stained with FITC anti-IgM (green), rhodamine anti-IgG (red), and 4',6'-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 µm. Original magnification, x20. C, Ig isotype-specific plasma cells (x10–5) in spleen populations of purified CD19highCD45R–/lowIgM+ and CD19highCD45R–/lowIgM– B cells, and unpurified splenocytes, as detected by Ig isotype-specific ELISPOT assays (mean ± SD; n = 4–6 independent cell samples/column). The numbers of IgM-, IgG1-, IgG2a-, IgG2b-, IgG3-, and IgA-secreting cells were 2337 ± 194, 494.5 ± 43.1, 358.3 ± 55.3, 399.5 ± 23.2, 0 and 274.5 ± 58.7 for CD19highCD45R–/lowIgM+ cells, 0, 2344.5 ± 581.6, 1194 ± 291, 949 ± 62, 454 ± 111.7, and 8800 ± 1414 for CD19highCD45R–/lowIgM– B cells, and 523 ± 60, 197.1 ± 131.3, 34.5 ± 3.5, 19 ± 9.9, 13.7 ± 2.1, and 157.5 ± 10.6 for total spleen cells. D, Frequencies of IgM-, IgG-, and IgA-secreting cells (x10–5) in unpurified splenocytes (gray histograms) and purified CD19highCD45R–/low spleen cells (black histograms) of infant mice at the indicated ages. Results are the mean ± SD of three independent experiments with splenocyte pools from animals of the same age.

 
IgM-secreting cells were already detected during the first days after birth, whereas switching to IgG and IgA isotypes took place at 5–7 days of extrauterine life (27). We tested whether the postnatal CD19highCD45R–/low B cells were also enriched in IgM-, IgG-, and IgA-secreting cells (Fig. 6D). As it happens in the adult, IgM plasma cells were moderately enriched among postnatal CD19highCD45R–/low B cells. They also included great frequencies of IgG-secreting cells (17.7-fold total spleen frequencies at day 7 vs 11.5-fold in the adult). By contrast, CD19highCD45R–/low B cells from infant mice showed a much lower enrichment in IgA plasma cells (6.7-fold increase over splenic cells) than that of the adult population, and they accounted for only a minor fraction of postnatal IgA-secreting cells (28). In particular, the contribution of the CD19highCD45R–/low B cell population to the vigorous expansion of IgA-secreting splenic cells occurring between days 15 and 30 postbirth was negligible.

The population of CD19highCD45R–/low mature B/plasma cells was preferentially generated from embryo-derived multipotential hemopoietic progenitors

The presence of CD19highCD45R–/low cells from very early periods in ontogeny and their persistence along life as a functionally distinct cell subset induced us to investigate their developmental origins. We performed in vitro differentiation and in vivo adoptive transfer experiments of multipotential hemopoietic progenitors from various ontogenic sources. Purified KSL multipotential progenitors from E11 and E15 FL, and from adult BM were established in ST-2 cell cultures, supplemented with IL-7 (Fig. 7A). When analyzed 3 and 6 days later, E11 progenitors gave rise to a single population of CD19+CD45R– cells. Only after maintaining these cultures for longer periods did a few CD19+CD45R+ cells appear (data not shown). E15 FL-derived KSL progenitors cultured in vitro produced both CD19+CD45R– and CD19+CD45R+ cell subsets. By contrast, adult BM-derived, multipotential progenitors were limited to the production of small amounts of CD19+CD45R+ cells. We also seeded E11-purified GFP+ KSL cells into GFP– FL organotypic cultures (16). When analyzed 14 days later, all the newly emerging GFP+ B-lineage cells displayed a CD19+CD45R– phenotype (data not shown).


Figure 7
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  		FIGURE 7. Regeneration of CD19highCD45R–/low and CD19+CD45R+ B cells with KSL hemopoietic progenitors from different ontogenical times. A, E11 and E15 FL, and adult BM purified KSL progenitors seeded in ST-2 plus IL-7 cultures, and analyzed 3 and 6 days later. The dot plots are from a representative experiment of four. Mean ± SD values of the CD19+CD45R– cells obtained in the 3 and 6 days cultures seeded with FL-derived KSL progenitors from E11 were 1.4 ± 0.5% and 23 ± 12% cells, respectively, and from E15 were 2.3 ± 0.9% and 36.5 ± 5%, respectively. CD19+CD45R+ cells obtained in the same E15 cultures were 6.9 ± 1.3% and 53.5 ± 5%, respectively, and in cultures seeded with adult BM-derived KSL progenitors were 1.7 ± 0.5% and 0.2 ± 0.1%, respectively. B, Representative examples of in vivo B cell repopulations obtained with the transfer of purified B6.CD45.1 KSL hemopoietic progenitors from 1) adult BM and 2) E11 FL into lethally irradiated, adult B6 hosts, and (3 ) from adult BM KSL cells into newborn, irradiated B6 hosts. Left histograms, Reconstitutions of CD45.1+ cells in the host spleens, and dot plots display the CD19/CD45R phenotype of the CD45.1+ cells. The numbers are the mean frequencies of CD19highCD45R–/low cells of donor origin. Right histograms, The CD45R level of donor-derived CD19+ B cells. C, Quantitative results of the in vivo reconstitutions of both CD19highCD45R–/low (left columns, graph) and CD19+CD45R+ B splenic cells (right columns, graph) derived from the transfers of purified KSL hemopoietic progenitors. The frequencies of splenic CD19highCD45R–/low cells were 0.5 ± 0.2% for adult B6.CD45.1 donors (1; n = 5) and 0.4 ± 0.2% for adult B6.GFP donors (2; n = 5). The frequencies of splenic CD19highCD45R–/low cells in total donor-derived cells present in transferred mice were 0.13 ± 0.07% for adult B6 recipients of adult B6.CD45.1 BM cells (3; n = 6); 0.1 ± 0.06% for adult B6 recipients of adult B6.GFP BM cells (4; n = 4); 1.3 ± 0.4% for adult B6 hosts of E11 B6.CD45.1 FL cells (5; n = 7); and 2.3 ± 0.7% for adult RAG-2/{gamma}–/– recipients of E11 B6.CD45.1 FL cells (6; n = 5); and 1.5 ± 0.5 for newborn B6 recipients of adult B6.CD45.1 BM (7; n = 4). The vertical axis represents the relative numbers of donor-derived cells of each population (mean ± SD). *, p < 0.01; **, p < 0.001.

 
In vivo adoptive cotransfers of KSL hemopoietic progenitors and congenic B6 BM cells were further performed. We established several donor/host experimental groups: B6.GFP+ cells into B6 recipient mice, B6.CD45.1 cells into B6 mice, and B6.CD45.1 cells into RAG-2/{gamma} chain–/– mice (14). CD45.1 donor-derived CD19highCD45R–/low B cells could be followed because, although lacking the CD45R isoform, they had a normal expression of the common CD45 leukocyte Ag (data not shown). We transferred KSL progenitors from E11 FL and from adult BM into adult and newborn, irradiated hosts (Fig. 7, B and C). The frequencies of donor-derived cell reconstitutions varied from 1.7% of total spleen cells for those derived from embryo sources up to 35.4% in the recipients of BM progenitors. The CD19+CD45R+ B cells were equally reconstituted in all experimental groups, and their frequencies were similar to those of normal controls. In contrast, the BM-derived KSL progenitors gave rise to low frequencies of the CD19highCD45R–/low B cells (p = 0.0013 and p = 0.0064, when compared with their frequencies in the donor B6.CD45.1 and B6.GFP spleens, respectively). E11 FL-derived KSL progenitors, however, reconstituted significantly more spleen CD19highCD45R–/low B cells in both B6 and RAG-2/{gamma} chain–/– recipients (p = 0.0022 and p = 0.0002, respectively, when compared with the CD19highCD45R–/low B cell reconstitutions obtained with BM CD45.1+ progenitors). Surprisingly, the transfers of BM-derived KSL progenitors into newborn mice also generated significantly higher frequencies of CD19highCD45R–/low B cells relative to the transfers of BM into adult hosts (p = 0.0025). We conclude that the adult population of spleen CD19highCD45R–/low mature B/plasma cells was preferentially regenerated by early embryofetal hemopoietic progenitors. It is also likely that cell-extrinsic signals acting in the early, perinatal stages of life play an additional role in the establishment of this B cell population.

Btk-deficient CBA/CaHN mice lack the splenic CD19highCD45R–/low B cell population

The early ontogenical origin of the CD19highCD45R–/low B cells and their bias to plasma cell differentiation were shared features with other innate-like B cells such as B1 and MZ B cells. Different mutations in BCR-signaling molecules selectively affect these lymphocytes, one of the best studied being Btk-deficient, xid mice (29). CBA/N (xid) mice lack B1 cells, although their spleens maintain normal levels of CD21highCD23– MZ B cells. When analyzed, the spleens of adult xid mice were completely devoid of the CD19highCD45R–/low B cell population (Fig. 8).


Figure 8
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  		FIGURE 8. Absence of CD19highCD45R–/low B cells from the spleen of adult CBA/CaHN.xid mice. Representative two-color CD19/CD45R staining of congenic CBA/CaJ and CBA/CaHN.xid spleens. The window defines the location of CD19highCD45R–/low B cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We describe here a novel subset of CD19highCD45R–/lowIgM+/–IgD–CD21–/lowCD23–/low B cells present in the spleen of normal, unimmunized mice, from very early periods after birth. The splenic CD19highCD45R–/low B cells exhibited a genetic program (low Pax-5 and high Blimp-1 and XBP-1 gene expressions) and surface phenotype (CD43, CD138, CD9) characteristic of activated, highly differentiated B cells (30). They were moderately enriched in IgM-secreting cells and included high frequencies of spontaneous IgG- and IgA-secreting cells, which represented up to one-quarter of total spleen plasma cells for some non-IgM isotypes (e.g., IgG2a and IgA). These cells were preferentially clustered in the periphery of primary follicles and in the MZ. A significant feature of the CD19highCD45R–/low cells is their low/negative expression of CD21/Cr2 and CD23 receptors. CD21 is up-regulated in spleen transitional B cells (31), provides BCR-costimulatory signals (32), and is involved in long-term B cell survival (33, 34). The lack of the CD21R does not seem to represent CD21 down-regulation linked to plasma cell differentiation, as this is an intrinsic feature of the population even in postnatal periods. It might represent an immaturity trait or a functional deficiency, but the CD19highCD45R–/low B cells were nevertheless able to produce high rates of IgG/IgA-secreting plasma cells. In fact, Cr2–/– mice also showed elevated titers of CD21-independent IgG plasma cells, which were efficient at rapidly coping with viral agents, although the responses were short-lived (35, 36). Immune B cells follow two complementary strategies in their responses against pathogens: 1) naive B lymphocytes bearing germline BCRs of moderate-to-low affinity to invading Ags require the concurrence of costimulatory signals and proceed into the GC reaction, where somatic hypermutation and affinity maturation mechanisms operate, and (2) B cells displaying evolutionarily selected, high-affinity BCRs for defined pathogen-associated patterns (such as highly repetitive, viral envelope determinants) do not rely on costimulatory signals and trigger rapid, although short-lived, extrafollicular responses to blood-borne pathogens (37). This latter type of immunity represents a first defensive barrier and includes cell types such as MZ and B1 B cells that predominantly, but not exclusively, secrete IgM and IgA Abs (4, 38, 39, 40). The splenic CD19highCD45R–/low B cells were relatively excluded from follicles, sitting in a niche (T cell-area/follicle border) shared with the earliest-produced Ig isotype-switched B cells in T cell-dependent immune responses (41), as well as with other extrafollicular lymphocytes. They may thus represent either a component of the early IgG responses and/or a GC-independent B cell subset; in agreement with the later possibility, CD19highCD45R–/low B cells remained as PNA– cells after SRBC immunization (our unpublished results). The common enrichment for both IgG- and IgA-secreting plasma cells inside the same CD19highCD45R–/low B cell subset was intriguing. Although FO B cells are biased to the production of IgG isotypes, other extrafollicular B cells, such as B1 and MZ B cells, have the propensity to produce natural IgM and to switch to IgA under minimal conditions, (40). In vivo, B1b cells were able to generate protective IgGs to pneumococcal polysaccharide (4). It remains the possibility that the CD19highCD45R–/low B cell population contains independent cell subsets each one producing either IgG or IgA Abs, but we have not been able to identify them. The IgA-secreting CD19highCD45R–/low cells could represent a postulated splenic counterpart of peritoneal B1b cells (42), themselves likely derived from the recently described BM CD19+CD45R– pre-B cells (12). Alternatively, IgG/IgA-secreting CD19highCD45R–/low spleen cells could be a physiological correlate of the distinct developmental pathway to IgG- and IgA-switched B cells described in Igµ-deficient mice, which is established at the level of B cell precursors and is T independent (43, 44). Although not induced by intentional immunization, the IgG/IgA production of the CD19highCD45R–/low B cells might be the result of regular microbial encounters (45), particularly, intestinal bacteria, but these cells also contributed importantly to the early postnatal IgG production, which is known to be developmentally controlled and (foreign) Ag independent (46). All together, the enrichment in Ig-secreting plasma cells, the extrafollicular location, and the negative/low expressions of CD45R and CD21 surface BCR coreceptors suggest a relationship of the splenic CD19highCD45R–/low cells with innate-like B cell types involved in rapid, costimulus-independent responses to frequent pathogens and/or to self-reactive Ags, specialized in natural IgG and IgA Ab production (7, 47). It was interesting to note that the CD19highCD45R–/low B cells were completely absent in Btk-deficient mice, further supporting the notion that, as it happens with B1 cells or MZ B cells, their generation may be conditioned by developmentally restricted BCR specificities that are selected by strong BCR-dependent signals (48). There is abundant information about the repertoires and the immunoregulatory/defense activities of natural IgM Ab responses (49), but much less is known about the significance of basal levels of IgG and IgA, generally considered to be secondary to foreign Ag stimuli. The genetic bases of both natural IgG and T cell-dependent IgG responses are distinct, as shown in STAT3–/– and CD19–/– mice (15, 50), and this fact suggests that both IgG types might proceed from functionally distinct compartments, as the one shown here.

The splenic CD19highCD45R–/low B cells were different from the described population of Ag-specific, post-GC CD19lowCD45R–CD11b+ preplasma cells, which were located in the red pulp, and whose myeloid character is under discussion (25, 51, 52, 53, 54). Also, in the hen egg lysozyme model of B cell autoreactivity, anergic self-reactive B cells were excluded from the follicle, switched to IgG by BCR-independent stimuli and displayed a surface phenotype partially shared with the one of CD19highCD45R–/low B cells (55). The novel B cell subset here described might also be reminiscent of the CD45R– IgG plasma cells that expanded in E- and P-selectin-deficient mice (45). In humans, a phenotypically similar CD5–CD45RAlow B cell subset mainly produced natural Abs of the IgM and IgA isotypes (56).

The detection of CD19highCD45R–/low cells with progenitor character in the fetus and in the adult BM, and of phenotypically similar B cells in the adult spleen, does not mean that all of these cells represent the same cell type or even that they are developmentally related. However, a frequent trait of many innate-like lymphocytes ({gamma}{delta} T, B1, and MZ B cells) is their emergence in the early ontogeny (7). In adoptive cell transfers, the main CD19+CD45R+ B cells were equally generated by both embryo and adult progenitors (although more slowly in the in vitro cultures of E11 FL). Whereas the adult recipients of E11 FL progenitors contained normal numbers of CD19highCD45R–/low B cells, very few of these latter were observed in the recipients of adult BM progenitors, suggesting that they had a highly reduced potential to differentiate into the CD19highCD45R–/low B cells. Interestingly, when inoculated into newborn recipients, adult KSL progenitors did give rise to CD19highCD45R–/low B cells, further implying that cell-extrinsic inductive signalings in perinatal life periods, which may be Ag specific, also stimulated the generation/selection of CD19highCD45R–/low cells from adult BM progenitors. During the course of this project, Montecino-Rodriguez et al. (12) reported a population of fetal and adult BM CD19+CD45R– B cell progenitors that reconstituted CD5+/–CD11b+ peritoneal B1b, but not B2, cells. We have not focused on peritoneal B1 cells, because their CD45Rlow phenotype was partially shared by the spleen CD19highCD45R–/low B cells and could have led to confusing conclusions, but we cannot discard that the splenic CD19highCD45R–/low mature B cells might represent another product of the FL/BM CD19+CD45R– B cell precursors, particularly devoted to natural IgG and IgA secretion. Those authors considered the developmental origin of the BM precursors, variously proposing either that they could be derived solely from fetal progenitors and later maintained by intrinsic self-renewal, or that they continuously differentiated from BM-derived multipotent progenitors (12). In our experiments, adult BM KSL progenitors showed a limited potential to generate the splenic CD19highCD45R–/low B cell population. If both the splenic mature B and BM B cell progenitor subsets are related, our findings would suggest that the adult BM CD19+CD45R– B cell precursors (or the mature splenic CD19highCD45R–/low B cells here reported) should maintain their population size, at least partially, by self-renewal, as has been proposed for B1 B cells, rather than being continuously derived from adult multipotent progenitors. Actually, CD19highCD45R–/low B cells were actively proliferating cells. These latter results might also contribute to understand and treat the specific B cell deficiencies occurring after transplantation of BM hemopoietic stem cells in humans, where lymphoid activities derived from progenitors of the early ontogeny may not be fully reconstituted, as has been described for IgG-secreting plasma cells (57, 58, 59).


   Acknowledgments
 
We thank A. Vicente, B. Palacios, and F. Martínez for excellent technical assistance; C. Moreno and D. Barber (Departamento de Immunología y Oncología, Centro Nacional de Biotecnología) for help with flow cytometry and for providing the CD19–/– mouse controls, respectively; C. Bellas (Hospital Puerta de Hierro), for advice on in situ immunofluorescence experiments; and P. Mason for editorial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Instituto de Salud Carlos III (ISCIII PI05/007, ISCIII 03/18) and by Ministerio de Educación y Ciencia (SAF2003-08194). We also acknowledge the intramural funding received by the Centro de Biología Molecular S.O. from the Ramon Areces Foundation and the Banco Santander Central Hispano. P.G., I.C., N.S., B.d.R., and B.d.A. were supported by ISCIII fellowships and by the Ramón y Cajal Program. Back

2 Address correspondence and reprint requests to Dr. Belén de Andrés, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain; E-mail address: bdandres{at}isciii.es or Dr. María Luisa Gaspar, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain; E-mail address: mlgaspar{at}isciii.es Back

3 Current address: Centro Nacional de Investigaciones Cardiovasculares, Instituto de Salud Carlos III, C/Melchor Fernández Almagro, 3, Madrid, Spain. Back

4 Abbreviations used in this paper: FO, follicular; GC, germinal center; MZ, marginal zone; FL, fetal liver; E, gestational day; BM, bone marrow; HPRT, hypoxanthine guanine phosphoribosyl transferase; KSL, c-Kit+Sca-1+Lin–. Back

Received for publication March 12, 2007. Accepted for publication August 7, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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