A New Population of Cells Lacking Expression of CD27 Represents a Notable Component of the B Cell Memory Compartment in Systemic Lupus Erythematosus

Clinical samples

Samples were obtained with informed consent using protocols approved by the University of Rochester Medical Center Institutional Review Board. PBMCs from SLE patients, hepatitis C patients, rheumatoid arthritis (RA) patients, and healthy controls were isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation (Pharmacia Biotech). Eligible SLE and RA subjects were male or female, ages >18 years, who met the American College of Rheumatology revised criteria for the classification of disease. Disease activity was assessed by the Systemic Lupus Activity Measure. Tonsil samples were obtained from normal subjects undergoing routine tonsillectomy.

Serological analyses

Autoantibodies in the sera against dsDNA were detected by ELISA using the Kallestad anti-dsDNA kit (Bio-Rad). Detection of Abs in the sera against RNP, Sm, Ro, and La were conducted using the QUANTA Lite Microwell ELISA kits (INOVA Diagnostics). C3 levels in the sera were quantitatively determined by rate nephelometry using the IMMAGE immunochemistry systems (Beckman Coulter). Assays for autoantibodies and C3 were conducted following the manufacturers’ recommendations, respectively, in the Department of Pathology and Laboratory Medicine Immunology Laboratory and Protein Laboratory at the University of Rochester.

Flow cytometry analysis

Experiments were performed as previously described in our laboratory (12, 14, 15). PBMCs purified through Ficoll-Hypaque density gradient centrifugation were stained in PBS/2 mM EDTA/0.5% BSA/5% normal mouse serum with fluorochrome-conjugated mouse monoclonal anti-CD19 (SJ25C1; BD Biosciences), anti-IgD (IA6-2; BD Biosciences), anti-CD27 (O323; eBioscience), and one of the following Abs: anti-CD38 (HIT2; BD Biosciences), anti-IgM (G20-127; BD Biosciences), anti-IgG (G18-145; BD Biosciences), anti-CD45R/B220 (RA3-6B2; eBioscience), or anti-CD10 (HI10a; BD Biosciences). FcRH4 was detected either with biotinylated goat anti-human FcRH4 (R&D Systems) followed by fluorochrome-conjugated streptavidin or with a mAb provided by Dr. M. D. Cooper (University of Alabama at Birmingham). Tonsillar B cells were first isolated by SRBC rosetting, then stained with anti-IgD, anti-CD27, anti-CD38, and one of the following Abs: anti-CD10, anti-IgG, anti-IgM, and anti-CD45R/B220. To analyze FcRH4 expression, tonsil cells were stained with anti-CD19, anti-IgD, anti-FcRH4, and anti-CD27 or anti-CD38. After washing, cells were fixed in PBS/0.5% formaldehyde and analyzed on a FACSCalibur (BD Biosciences). To analyze the extrusion of Rhodamine 123 (R123) by different subsets of B cells, PBMCs or tonsillar mononuclear cells were pulsed with 6 μM R123 (Molecular Probes) in the culture medium at 37°C for 10 min, then chased for 3 h. After washing, cells were stained with anti-CD19, anti-IgD, and anti-CD27 at 4°C for 30 min as described above.

To carry out nine-color flow cytometry, PBMCs or tonsillar mononuclear cells were stained with the following fluorochrome-conjugated mouse anti-human mAbs: FITC-anti-IgD (IA6-2; BD Biosciences), PE-anti-CD38 (HIT2; BD Biosciences), PE-Alexa 610-anti-CD24 (SN3; Caltag Laboratories), PE-Cy5-anti-IgM (G20-127; BD Biosciences), PerCP-Cy5.5-anti-CD138 (MI15; BD Biosciences), PE-Cy7-anti-CD10 (HI10a; BD Biosciences), Pacific Blue-anti-CD3 (SP34-2; BD Biosciences), allophycocyanin-anti-CD27 (O323; eBioscience), and allophycocyanin-Cy7-CD19 (SJ25C1; BD Biosciences). A set of Simply Cellular Compensation Standard beads (Bangs Laboratories), each stained with one of the above Abs, was set up as compensation controls. A set of fluorescence-minus-one controls was also included to facilitate the gating and differentiation of positive from negative populations.

For our studies, memory cells were either identified on the basis of CD27 expression or according to their Bm5 phenotype within the Bm1–Bm5 classification (Bm1: IgD⁺CD38⁻; Bm2: IgD⁺CD38^dull; Bm2′: IgD⁺CD38⁺; Bm3–Bm4: IgD-CD38⁺; early Bm5: IgD⁻CD38^dull; and Bm5: IgD⁻CD38⁻) (16, 17).

Proliferation assay

Peripheral or tonsillar B cells were negatively selected using the B cell Isolation Kit II (Miltenyi Biotec). Tonsillar B cells were further depleted of CD10⁺ and CD27⁺ cells with MACS magnetic selection (Miltenyi Biotec). This population was further fractionated on the basis of IgD expression into CD10⁻CD27⁻IgD⁺ (naive) and CD10⁻CD27⁻IgD⁻ (DN) populations with magnetic beads (Miltenyi Biotec). Isolated cells were first loaded with 5 μM CFSE (Molecular Probes) in PBS/0.1% BSA at 37°C for 10 min, then cultured for 4 days in RPMI 1640 medium containing 10% FCS, 10 ng/ml IL-2, and IL-10 (both from PeproTech) and stimulated with 2.5 μg/ml CpG2006 oligonucleotide (Oligos Etc.) in the presence or absence of 2.5 μg/ml goat F(ab′)₂ anti-human IgM (Jackson ImmunoResearch Laboratories). After culture, cells were stained with anti-IgD and anti-CD27 for FACS analysis.

V gene sequence analysis

PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and stained with FITC-IgD, PE-IgG, PerCP-CD20, allophycocyanin-CD27, and allophycocyanin-Cy7-CD19. CD19⁺CD20⁺IgG⁺IgD⁻ cells were sorted into CD27⁺ and CD27⁻ populations using a FACSAria (BD Biosciences). Total RNA was extracted from the sorted cells and reverse transcribed into cDNA using oligo(dT) priming, which was then amplified by PCR with a V_H3 family-specific primer (5′-GAGGTGCAGCTGKTGGAGTCTGG-3′) and a Cγ primer (5′-CGGTTCGGGGAAGTAGTCCTTGACC-3′) that is identical among all of the IgG isotypes. The PCR products were then subcloned into the pCR4Blunt-TOPO vector (Invitrogen Life Technologies) and used for the transformation of competent DH5α Escherichia coli cells. After transformation, plasmid DNA was isolated for sequencing from randomly picked colonies. Sequencing was conducted with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) using the M13F primer (5′-GTAAAACGACGGCCAGT-3′) and analyzed in an automated Applied Biosystems sequencer.

Statistical analysis

Statistical significance was assessed by the two-tailed Student’s t test assuming unequal variances. The frequencies of clinical manifestations and autoantibodies between groups were compared by a χ² test of independence.

A population of peripheral blood B cells lacking expression of CD27 and IgD is expanded in patients with SLE

When analyzed for their expression of surface IgD and CD27, human PBL B cells typically segregate into two dominant populations as follows: 1) an IgD⁺CD27⁻ fraction, which largely includes mature naive B cells, and which generally represents the main population in healthy subjects and 2) a fraction containing CD27⁺ memory B cells, which can be further separated into IgD⁺ and IgD⁻ subsets (Fig. 1⇓A). As described by multiple groups, patients with SLE frequently have a relative expansion of CD27⁺ memory cells. In addition, Davidson’s laboratory and our own group have reported that some SLE patients also display an expansion of CD19⁺ IgD⁻ CD27⁻ (DN) B cells (Fig. 1⇓A) (11, 12). As shown in Fig. 1⇓A and in Table I⇓, in some patients, this fraction constitutes the largest PBL B cell population.

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FIGURE 1.

IgD/CD27 DN B cells are expanded in SLE. A, Distribution of peripheral blood B cell subsets identified by their surface expression of IgD and CD27. Representative examples of the profiles observed in healthy subjects and SLE patients with expansion of DN cells are shown. The numbers attached to the corresponding quadrants represent the percentage of the annotated subset among all CD19⁺ live lymphocytes. N, Naive; NSM, nonswitched memory; SM, switched memory; PC, plasma cells. B, The relative abundance of DN cells within CD19⁺ PBL is shown for different cohorts of patients. An increased frequency of DN cells is found in 50% of SLE patients (SLE^high). The frequency of these cells was significantly higher in SLE than in healthy subjects, patients with RA, and patients with chronic hepatitis C (HepC). Statistical values, comparison between normal values and different diseased cohorts. No significant difference was found among normal subjects, RA, and hepatitis C.

We have now analyzed this DN population in 36 patients with SLE, as well as in normal subjects and diseased controls. As a group, the frequency of DN cells among total PBL B cells is significantly increased in SLE patients as compared with healthy controls (p = 0.0001). In healthy subjects, DN cells are always lower than 10% of all CD19⁺ B cells, with a mean frequency of 4.5% (Table I⇑ and Fig. 1⇑B). In contrast, DN cells represent >10% of CD19⁺ B cells in 50% (18 of 36) of SLE patients, with a mean frequency for this subgroup of 19.4%. In 14% (5 of 36) of patients, DN cells reached levels higher than 20% of all PBL B cells. Moreover, DN cells were present at frequencies higher than CD27⁺ memory cells in 25% of all SLE patients analyzed and in 40% of the SLE subset classified as having DN expansions (Table I⇑). As illustrated in Table I⇑, the relative increase observed in the frequency of DN cells was not solely due to the naive lymphopenia that can be observed in SLE patients (18). Indeed, the frequency of DN cells was increased in 72% (13 of 18) of patients in whom the frequency of naive B cells fell within the normal range for healthy controls.

To assess whether DN cells are also expanded in other conditions characterized by autoimmunity and/or B cell hyperactivity, we studied patients with RA and patients with chronic hepatitis C infection. As shown in Fig. 1⇑B, the frequency of DN cells in these diseases was no different from the one observed in healthy subjects.

Surface phenotype of CD27/IgD DN B cells in human PBL

The surface phenotype of PBL DN cells resembles that of conventional CD27⁺ memory cells, both in SLE patients and healthy controls (Figs. 2⇓ and 3⇓). Thus, DN cells contain similar frequencies of IgG⁺ isotype-switched cells (44.4 ± 13.0% for healthy controls and 53.7 ± 20.5% for SLE) as CD27⁺ cells (42.8 ± 10.8% and 43.2 ± 10.1%, respectively). The frequency of IgM⁺ cells was also similar between DN cells (14.6 ± 8.0% in healthy controls vs 14.8 ± 7.1% in SLE) and CD27⁺ cells (17.5 ± 9.1% vs 20.6 ± 11.8%, respectively). It should be noted, however, that in contrast to DN cells, the vast majority of CD27⁺ memory cells that express IgM also express IgD (data not shown) (7, 19).

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FIGURE 2.

Surface phenotype of peripheral blood DN B cells. A, Dot plots represent staining of PBL B cells in healthy subjects and SLE patients with increased frequency of DN cells. The histograms demonstrate the expression of IgG, CD38, and B220 on naive, CD27⁺ memory, and DN cells. DN cells and CD27⁺ memory cells display virtually identical phenotypes. Of note, in SLE, both CD27⁺ memory cells and DN cells are almost universally CD38^dull (eBm5 or early Bm5) at the expense of the CD38-negative fraction typically found in normal samples (Bm5). B, Multiparameter analysis of DN B cells. Healthy PBL lymphocytes (from a side scatter (SSC)-A vs a forward scatter (FSC)-A gate) were analyzed using eight-color flow cytometry for the expression of CD3, CD19, CD27, IgD, IgM, CD10, CD24, and CD38. The contour plots to the right show two-dimensional plots to compare the expression of several markers between DN cells and conventional subsets defined by IgD and CD27 staining. These results further illustrate the similar surface phenotype of CD27⁺ cells and DN cells, including the presence of CD24⁺ and CD24⁻ populations in the isotype-switched memory subset and in the DN subset (which, in this particular example, was predominantly isotype switched). CD27⁺ memory cells and DN cells also showed significant differences with naive B cells when the latter subset is conventionally defined as IgD⁺CD27⁻. Thus, naive B cells display lower expression of CD24 and typically lower expression of surface IgM than unswitched CD27⁺ memory cells. The former subset also includes a small population of CD24²⁺CD38²⁺ that corresponds to transitional B cells also expressing CD10 (23 ,24 ). Of note, isotype-switched CD27⁺ and DN cells also contain a small fraction of CD10⁺ cells that likely represent recirculating pre-GC cells (22 ).

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FIGURE 3.

Multichromatic analysis of tonsil DN B cells. Healthy tonsil B cells were analyzed using the same eight-color protocol as in Fig. 2⇑B. Upper right dot plots, conventional classification obtained by analysis of IgD/CD27 (N, naive; NSM, nonswitched memory; SM, switched memory; PC, plasma cells) and IgD/CD38 (Bm1–Bm5; plasma cells). In contrast to PBL, in tonsils both CD27⁺ cells and IgD⁻CD27⁻ cells contain a CD38⁺CD10⁺ fraction that represents GC cells as well as a CD38^neg-dull fraction that lacks CD10 expression. In contrast, Bm5 cells are devoid of CD10⁺ GC cells due to the exclusion of cells expressing higher levels of CD38 but can be divided into CD27⁺ and CD27⁻ fractions. Thus, the CD27⁻IgD⁻CD10⁻ fraction represents the tonsil equivalent of PBL DN cells. Further analysis of CD27⁺ switched memory and DN cells reveals a virtually identical profile regarding the expression of CD24 and IgM. This identity was found whether DN were identified within CD27⁻IgD⁻ or Bm5 fractions.

DN cells are also similar to CD27⁺ memory cells in terms of their level of expression of CD38 (Figs. 2⇑ and 3⇑). Indeed, DN cells include CD38^dull and CD38⁻ populations whose levels of CD38 parallel those expressed by early Bm5 and Bm5 memory cells, respectively, and below those of B cells that typically stain clearly positive for this marker in the PBL (pre-GC, transitional, and plasmablasts) (17). Interestingly, although Bm5, CD27⁺, and DN cells are almost equally split into CD38⁻ and CD38^dull in healthy subjects, in SLE patients these populations are largely formed by the CD38^dull subset (early Bm5). Also, as previously described by Bleesing and Fleisher (20) and our own group (21) and in contrast to naive B cells, which universally express B220, conventional memory cells can be divided into B220-positive and -negative subsets. This heterogeneity is also found in DN cells.

It is worth bearing in mind that classification schemes of human B cells based on the expression of IgD, CD27, and/or CD38, although useful and widely used, have significant limitations. Thus, so-called naive Bm1 and Bm2 cells also contain non-switched CD27⁺ memory cells; Bm2′ pre-GC cells also contain transitional B cells, which can also be found within the IgD⁺CD27⁻ naive populations (22, 23, 24). We therefore used multichromatic flow cytometry to evaluate the expression of other useful differentiation markers including CD24 and CD10 (Figs. 2⇑B and 3⇑). This approach confirms the similarity in surface phenotype between DN and conventional CD27⁺ memory cells. Furthermore, it shows that DN cells are devoid of surface CD10, a feature that adds further to the distinction between DN cells and other B cell subsets that can be found in the PBL, including transitional cells (CD10⁺, CD38^high, IgD⁺) and pregerminal center (CD10⁺, CD38^high, IgD^+/−;15, 17, 23, 24, 25, 26).

Surface phenotype of CD27/IgD DN B cells in human tonsil

Next, we sought to identify the tonsil counterpart of the DN population. As shown in Fig. 3⇑, IgD⁻CD27⁻ tonsil B cells can be further divided into CD38⁺ and CD38^dull-neg. The former fraction stains positive for CD10 and, as previously shown by multiple groups, it represents GC cells, whereas the latter subset lacks expression of CD10 and represents the tonsil counterpart of DN cells (15, 17, 23, 25, 26). As previously discussed for the PBL, the Bm1–Bm5 classification has significant limitations and, as shown in Fig. 3⇑, the Bm5 subset contains not only conventional CD27⁺ memory cells, but also a large fraction of CD27⁻ cells. We therefore compared the extended surface phenotype between the global IgD⁻CD27⁻CD10⁻ fraction, the Bm5 IgD⁻CD27⁻CD10⁻ fraction, the Bm5 IgD⁻CD27⁺CD10⁻ fraction, and the CD10⁻ fraction of IgD⁻CD27⁺ memory cells. Remarkably, all four subsets display a virtually identical phenotype with regard to the expression of CD24 and IgM. Similarly, all fractions are comparable in terms of their expression of IgG and B220 (data not shown).

Another important difference between PBL and tonsil DN cells is determined by the expression of FcRH4, an interesting inhibitory FcR homolog that has been described during the preparation of this manuscript as being present in a subset of tonsil CD27neg memory cells, but absent in the PBL, bone marrow, and spleen (13, 27). As shown in Fig. 4⇓, and consistent with the results of Ehrhardt et al. (13), FcRH4 expression is largely restricted to a fraction of CD27neg cells, whether IgD⁺ or isotype switched. Tonsil CD27neg cells, however, include a significant component of GC cells. Therefore, we assessed the expression of FcRH4 in the tonsil DN counterpart by analyzing the Bm5 CD27neg population. This approach demonstrates that tonsil DN cells contain a majority of FcRH4⁺ cells as well as a fraction that lacks expression of this marker. In contrast, virtually no expression of FcRH4 was detected in PBL DN cells, whether in healthy subjects or in SLE patients.

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FIGURE 4.

Peripheral blood DN B cells lack expression of FcRH4. PBMC were stained for CD19, IgD, CD27, and FcRH4, whereas purified tonsil B cells were stained for IgD, CD27, CD38, and FcRH4. A, Healthy PBL; B, SLE PBL from a patient with increased frequency of DN cells; and C and D, normal tonsil. Expression of FcRH4 is absent from all B cell subsets in the PBL of both SLE and healthy subjects. In contrast, FcRH4 expression is readily detected in a fraction of tonsillar IgD⁻CD27⁻ cells and Bm5 cells.

CD27/IgD DN B cells have phenotypic, genetic, and functional characteristics of memory cells

As previously discussed, with the critical exception of CD27, the surface phenotype of DN cells strikingly resembles that of conventional memory cells, whether they are identified through the expression of CD27 or by the Bm5 classification. Several additional features permit such characterization. Thus, as shown in Table II⇓ and Fig. 5⇓, DN cells display a significant level of somatic hypermutation in their rearranged V_H Ab genes. Interestingly, this level was not significantly different between healthy controls and SLE patients (means of 3.2 vs 2.6%, respectively). However, the mutation level displayed by CD27neg cells is lower than the mutation rate observed in their CD27⁺ counterparts (5.4% in normal subjects and 5.1% in SLE). Overall, the distribution of mutations and the replacement:silent mutation ratio suggests that DN cells are Ag experienced and may have been selected by an Ag-driven process.

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FIGURE 5.

V_H genes from CD27⁻IgG⁺ memory B cells exhibit fewer somatic mutations than those from CD27⁺IgG⁺ memory B cells. CD27⁻IgG⁺ (□) and CD27⁺IgG⁺ (▪) memory B cells were sorted from the periphery of a healthy donor (A) and a SLE patient (B), and their rearranged V_H sequences were analyzed. The percentages of the total V gene sequences exhibiting certain numbers of mutations per V gene sequence are plotted.

As recently described by others, memory B cells are also characterized by their inability to extrude rhodamine or similar dyes owing to the absence of the ATP-binding cassette B1 transporter (28). As shown in Fig. 6⇓, this property is shared by DN cells and conventional CD27⁺ memory cells, with both subsets showing a significant departure with the behavior of naive B cells. Finally, we determined whether DN cells behave like conventional memory cells with regard to their proliferative behavior in response to stimulation with CpG DNA. As shown in Fig. 7⇓, both populations show a similar positive response in the absence of BCR costimulation. This type of response further differentiates DN cells from naive B cells, the latter of which require BCR costimulation for effective proliferation (29). Of note, CD27 gets up-regulated by the majority of DN cells that proliferate in response to CpG DNA stimulation.

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FIGURE 6.

DN cells fail to extrude R123. PBMC from a healthy subject and a lupus patient and tonsil mononuclear cells from a healthy subject were stained for surface expression of IgD and CD27 and their intracellular content of R123. Red histograms represent IgD⁺CD27⁻ cells (naive and transitional which stain negative and positive, respectively, for R123). CD27⁺ memory cells and DN cells (green and black histograms, respectively) demonstrate similar positive R123 staining.

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FIGURE 7.

Proliferation of DN cells in response to CpG DNA stimulation. A, As compared with naive B cells, DN cells show a significantly greater degree of proliferation in response to CpG DNA in the absence of BCR cross-linking. B, Upon stimulation with CpG DNA (in the presence or absence of BCR cross-linking), proliferating DN cells up-regulate CD27 expression.

Clinical correlations

To identify correlations between increased frequency of DN cells and clinical manifestations of SLE, we studied 46 patients that were classified as either DN-low (DN <10% of all PBL B cells; n = 20) or DN-high (DN ≥ 10%; n = 26). As compared with DN-low patients, DN-high patients had a significantly higher frequency of nephritis (15 of 26 vs 4 of 20; p = 0.025). Histology was available in three of four patients with nephritis in the DN-low group and in 12 of 15 patients in the DN-high group. Classes III and V (30) were predominant in this group, as they were present in six and five patients, respectively (of these patients, one had classes III plus V and another classes IV plus V). One additional patient had type II nephritis. In the DN-low group, classes III, IV, and V were diagnosed in one patient each.

DN-high patients were also characterized by an increased titer of anti-dsDNA Abs (p = 0.001) and anti-RNP/Sm Abs (p = 0.009). No significant differences were observed between the DN-high and DN-low groups in terms of hematological manifestations, anti-Ro/La Abs, and serum C3 levels. In a subset of 23 patients for whom Systemic Lupus Activity Measure values were available, significantly higher scores were found in the DN-high group (p = 0.02). Interestingly, DN-high patients also had significantly higher levels of serum 9G4 Abs, an autoantibody species with high specificity for SLE, which has been shown to correlate with disease activity and lupus nephritis (21, 31, 32). For patients whose DN cells reached levels >20% of all PBL B cells, neither clinical nor serological profiles were significantly different from those of patients with DN levels between 10 and 20%.