B cell co-receptors regulating T cell-dependent antibody production in common variable immunodeficiency: CD27 pathway defects identify subsets of severely immuno-compromised patients

Serge Jacquot, Laëtitia Maçon-Lemaître, Estelle Paris, Tetsuji Kobata1, Yuetsu Tanaka2, Chikao Morimoto3, Stuart F. Schlossman3 and François Tron INSERM U519, IFRMP 23, Laboratoire d'Immunopathologie Clinique et Expérimentale, CHU Charles Nicolle, Faculté de Médecine et de Pharmacie, 22 boulevard Gambetta, 76183 Rouen Cedex, France
1 Division of Immunology, Institute for Medical Science, Dokkyo University School of Medicine, Tochigi 321-0293, Japan
2 Department of Infectious Disease and Immunology, Okinawa-Asia Research Center of Medical Science, University of the Ryukyus School of Medicine, Okinawa 903-0215, Japan
3 Division of Tumor Immunology, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA

Correspondence to: S. Jacquot


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD27 and CD134 ligand (CD134L) are two B cell co-receptors for Th cell activation-induced ligands (i.e. CD70 and CD134) that promote differentiation of B cells into plasma cells and high-rate antibody production respectively. We explored the CD27 pathway and T cell CD134 expression in common variable immunodeficiency (CVID), a disease characterized by a lack of plasma cells and low Ig serum levels. Twelve patients were compared to seven healthy controls. We found a low percentage of circulating CD27+ B cells in seven patients and B cell CD27 expression was not up-regulated by in vitro activation in two of them. Importantly, the number of circulating CD27+ B cells was correlated with the severity of the disease—the patients with the lowest CD27+ B cell counts having the lowest serum Ig concentrations and the lowest total peripheral blood B cell counts. In contrast, CD70 and CD134 were normally expressed on in vitro activated T cells. CD134L was not detected on patient and control B cells in our activation conditions. Functional studies of in vitro Ig production demonstrated an absence of B cell response to CD27 cross-linking, in particular in a patient with normal CD27 expression. Our results indicate that a defect in CD27 expression or function contributes to the pathogenesis of certain severe forms of CVID.

Keywords: memory B cells, T-B interactions, tumor necrosis factor receptor family


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Common variable immunodeficiency (CVID), the most frequent symptomatic primary antibody deficiency syndrome in humans, is characterized by a marked reduction in Ig serum levels, recurrent bacterial infections, and increased occurrence of autoimmune diseases and malignancies (1). Patients have low to normal numbers of circulating B cells with frequent defects in their capacity to differentiate in response to various stimuli. T cell dysfunction has also been shown in CVID, in particular a decreased production of IL-2, IL-4, IL-5 and IFN-{gamma} (2). However, identification of a precise molecular basis for the disease remains so far elusive and it is likely that CVID is a syndrome including different diseases (3). Nevertheless, common features in CVID are defects in terminal B cell differentiation resulting in a lack of plasma cells in tissue sites.

B cell activation and differentiation depend upon cognate T cell–B cell interactions involving several ligand–receptor pairs on both cell types and cytokine production. We and others have previously demonstrated that interaction between the B cell co-receptor CD27 and its ligand, CD70, on activated Th cells plays a critical role in T cell-dependent B cell differentiation into plasma cells (49). Moreover, CD27 was recently identified as a marker of memory B cells (10,11), a B cell subset prone to differentiation into plasma cells (12,13). CD27 is a member of the tumor necrosis factor (TNF) receptor family (14). This family includes other important molecules controlling B cell fate that can promote either differentiation towards germinal center cells and memory B cells such as CD40 (15), apoptosis such as CD95 (16) or survival such as BCMA (B cell maturation antigen) (17). In addition to CD27, we recently found that CD134 ligand (CD134L), a member of the reciprocal family of ligands related to TNF, after cross-linking with CD134 expressed on activated T cells, also delivers to B cells signals enhancing antibody production in humans (18) as previously shown in mice (19,20). These observations prompted us to investigate the CD27–CD70 and CD134L–CD134 pathways in CVID. Our results do not provide evidence for an abnormal regulation of CD134 expression on patient T cells. In contrast, we show here that CD27 is poorly expressed or not functional on B cells of a subset of CVID patients presenting a severe immunodeficiency, whereas their T cells have normal CD27 expression and high or normal CD70 expression.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients and controls
Twelve CVID patients were studied (Table 1Go). All patients had a history of recurrent bacterial infection of the upper and lower respiratory tracts, and all of them were treated with monthly i.v. Ig infusions. Seven healthy volunteers (age 24–40 years) were also analyzed. Blood samples were obtained after informed consent from patients and controls.


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Table 1. Characteristics of CVID patients
 
mAb
The following fluorochrome-labeled mAb were obtained from commercial sources: CD3 (UCHT1, IgG1), CD19 (B4, IgG1), CD27 (1A4, IgG1) and CD70 (2F11, IgG1) (Coulter, Miami, FL), and CD134 (ACT35, IgG1) (PharMingen, San Diego, CA). The 5A8 anti-CD134L (IgG1) (18) and the CD3 (OKT3, IgG2a) (21) mAb were produced locally.

Cell isolation and culture
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation and depleted of monocytes by adherence to the plastic surface of culture dishes. For some experiments, B cells were further purified by positive magnetic selection with CD22 mAb-coated beads (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's instructions. More than 95% of the cells obtained expressed CD19 as assessed by flow cytometry. PBMC (106/ml) were cultured at 37°C with 5% CO2 and 95% humidified air in 12-well plates (Costar, Cambridge, MA) coated with CD3 mAb in RPMI 1640 supplemented with 10% FCS and antibiotics (Life Technologies, Gaithersburg, MD). The kinetics of cell-surface molecule expression (CD27, CD70 and CD134) during lymphocyte activation was followed by flow cytometry from day 0 to 5.

Flow cytometry
Cells were labeled with FITC- or phycoerythrin (PE)-conjugated mAb and flow cytometry analysis was performed on an Epics Elite flow cytometer (Coulter, Hialeah, FL). The lymphocyte population was identified in forward/sideward scatter and fluorescence intensity data were collected on 104 gated lymphocytes. Isotype-matched mouse IgG (Coulter) negative controls were used throughout the studies and always reacted with <5% of the cells.

In vitro IgG production and quantification by ELISA
For some patients and controls, purified B cells (106/ml) were cultured in triplicates in 96-well round-bottom plates in 0.2 ml of culture medium with formalinized Staphylococcus aureus Cowan I strain (SAC) (Pansorbin; Calbiochem, San Diego, CA) (1:5000, v/v) and IL-2, 30 U/ml. The previously described (22,23) CD70 cDNA-transfected cells, CD70/300-19, or vector alone-transfected cells, mock/300-19, were added to the culture (2.5x104 cells/well) to provide CD27 cross-linking or as a negative control respectively. The CD70/300-19 and mock/300-19 cell lines were maintained in culture medium supplemented with G418 (Life Technologies) and fixed with 1% paraformaldehyde in PBS for 5 min before being added to the B cell culture.

After 7 days, culture supernatants were harvested and added to goat anti-human Ig (Caltag, Burlingame, CA)-coated 96-well flat-bottom ELISA plates. After discarding supernatants and washing with 0.05% Tween 20 in PBS, the bound IgG were detected with biotinylated goat anti-human IgG (Caltag) at a dilution of 1/10,000), followed by addition of alkaline phosphatase-labeled streptavidin (1/3000) and p-nitrophenyl phosphate (Sigma, St Louis, MO) substrate. The amount of bound IgG was assessed by spectrophotometric analysis at 405 nm.


    Results
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 Abstract
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 Methods
 Results
 Discussion
 References
 
Reduced expression of CD27 on circulating B cells of a subgroup of CVID patients
CD27, a critical differentiation marker acquired during peripheral B cell maturation and driving differentiation into plasma cells, identifies the memory B cell population, which constitutes ~30% of circulating B cells in healthy adults. The percentage of peripheral blood CD19+ B cells expressing CD27 was measured by two-color flow cytometry in all patients and controls (Fig. 1Go). Seven out of 12 patients had a percentage of CD27+ B cells lower than controls (below the mean value – 1 SEM observed in the control group), equal or close to 0% for two of them, whereas the other five patients had CD27+ B cell levels within the normal range (Fig. 2Go). It is worth noting that all patients showed normal levels of circulating CD27+ T cells (data not shown and Fig. 1Go), ruling out a constitutive defect hampering the CD27 protein synthesis.



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Fig. 1. Circulating CD27+ B cells in a control subject and two CVID patients representative of the 11 patients tested. PBMC were stained with FITC–CD19 and PE–CD27 mAb, and analyzed by flow cytometry as indicated in methods. Representative histograms are shown for a control subject (A), and patients BG (B) and JFQ (C), who present reduced or normal circulating CD27+ B cell populations respectively. Delineation of CD27+ cells according to isotype control staining is shown on CD19+ gated lymphocyte for the control subject (A): upper histogram, isotype control; lower histogram: CD27 staining.

 


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Fig. 2. Percentage of CD27+ B cells in CD19+ gated lymphocytes. PBMC from all patients and controls were stained with FITC–CD19 and PE–CD27 mAb, and analyzed by flow cytometry as indicated in methods.

 
Importantly, patients with low CD27+ B cell percentages had the most severe clinical appearance, with B cell lymphopenia and dramatically reduced serum IgG levels at the time of diagnosis (Table 1Go).

Since in vitro activation can induce CD27 expression on B cells of normal individuals (4), we examined the kinetic of CD27 on activated B cells of CVID patients, in particular of those with reduced numbers of CD27+ B cells. PBMC were cultured on CD3 mAb-coated plates, leading to B cell stimulation by CD3-activated T cells, as previously described (24). No increase of CD27+ B cells was observed in patients AK and BG, who had the lowest CD27+ B cell percentages (Fig. 3Go). However, flow cytometric analysis still showed viable B cells in PBMC cultures from these two patients. Patient DG could not be tested. All the other four patients with a low circulating CD27+ B cell population and patients with normal numbers of CD27+ B cells showed a kinetic profile of CD27 up-regulation on B lymphocytes similar to that observed in the seven healthy controls (Fig. 3Go).



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Fig. 3. Expression of CD27 on in vitro activated B cells. PBMC were cultured from day 0 to day 5 on CD3-coated plates to stimulate B cells by CD3-activated T cells. The percentage of CD27+ B cells in CD19+ gated lymphocytes measured in flow cytometry is shown for patients AK ({circ}), JPM (•), AP ({triangledown}) and a representative individual of the seven healthy controls ({blacktriangledown}).

 
Normal or increased CD70 expression and normal CD134 expression on in vitro activated T cells of CVID patients
Next, we asked whether the expression pattern of the CD27 ligand, CD70, on resting and activated T cells is similar in CVID patients and controls. CD70 was not expressed on resting T cells in all patients and controls. The kinetic study of CD70 expression on CD3-activated T cells was performed in all patients and controls, and showed that patient T cells expressed CD70 at similar or higher proportions than control T cells (Fig. 4AGo).




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Fig. 4. Expression of CD70 (A) and CD134 (B) on in vitro activated T cells. PBMC were cultured from day 0 to day 5 on CD3-coated plates. The percentage of CD70+ (A) and CD134+ (B) T cells in CD3+ cells was measured by flow cytometry in all patients and controls. Results are shown as mean ± SEM for patients ({square}) and healthy controls (•).

 
CD134 is another activation-induced T cell surface molecule promoting Ig secretion by plasma cells (18). All patient T lymphocytes displayed the same kinetics of CD134 expression as all control T cells (Fig. 4BGo). CD134L, the CD134 counter-receptor on B cells, could not be induced in our activation conditions on B lymphocytes from controls and patients as well (data not shown).

Absence of response to CD27 cross-linking of B cells from certain CVID patients
Since we did not find a lack of CD70 or CD134 expression on activated T cells that could pinpoint for a defective Th cell function, we considered that, so far, the major defect observed is a decreased B cell CD27 expression in more than half of CVID patients. To further investigate possible CD27 defects in CVID, we looked for a functional abnormality of the CD27 signaling pathway and measured the in vitro Ig production in response to CD27 engagement. Purified B cells from four representative CVID patients with various status CD27 expression were activated with SAC and IL-2 in presence of either CD70/300-19 cells expressing CD27 ligand or mock/300-19 as a negative control. In all experiments, the efficiency of the CD27 cross-linking by CD70-transfected cells was checked on B cells obtained from normal individuals (Fig. 5Go). As expected, there was no response to CD27 engagement of B cells obtained from patient BG that fail to express CD27 in vivo or after in vitro activation. B cells from patient OD, although able to up-regulate CD27 after in vitro stimulation despite a low baseline level, did not respond to CD27 signaling either (data not shown). Finally, two situations were observed in patients with normal CD27+ B cell percentage (Fig. 5Go): in patient FP, Ig production was not increased by CD27 cross-linking, whereas in patient JCT, a response similar to normal controls was observed.



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Fig. 5. Effect of CD27 engagement on in vitro Ig production. Purified B cells from a healthy control and three representative patients with different profiles of CD27 expression (no expression on B cells, BG; normal expression, FP and JCT) were cultured in triplicates in the presence of SAC, IL-2 and CD70/300-19 cells (open columns) or mock/300-19 cells (filled columns). The IgG concentration was measured by ELISA as described in Methods in the supernatants of three different wells.

 
Taken together, our results suggest that defective CD27 signaling is frequent in CVID.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B cell responses are driven by T lymphocytes through cell contacts and cytokine production. We recently identified two B cell co-receptors for T cell activation-induced ligands, CD27 and CD134L, leading to plasma cell differentiation and high-rate antibody production respectively (6,18). Thus, we decided to investigate the expression and function of these two receptor–ligand pairs in CVID, a disease characterized by defective antibody responses and absence of plasma cell differentiation. We found normal induction of CD70 and CD134 on in vitro activated T cells but showed that B cell CD27 expression or function are impaired in a subset of CVID patients.

Our report confirms and extends results recently published by Brouet et al. when we were writing this manuscript (25). CVID patients can indeed be separated in two groups, one with a normal number of circulating CD27+ B cells and one with a decreased, sometimes markedly, number of CD27+ B cells. Moreover, we showed that patients with a low CD27+ B cell pool have a more severe immunodeficiency with profound hypo-{gamma}-globulinemia at the time of diagnosis and reduced number of B cells, the later being associated with higher mortality in CVID (26). CD27 is an important differentiation marker acquired in secondary lymphoid organs during the process of peripheral B cell maturation in response to antigens and the absence of CD27+ B cells may indicate an impairment of peripheral B cell maturation contributing to this severe form of CVID. Interestingly, Lévy et al. reported that a subgroup of CVID has a defect in Ig VH gene somatic hypermutation (27). It will be important to determine whether these patients correspond to those displaying a reduced CD27+ B cell pool. The precise sequence of molecular events inducing somatic hypermutation or up-regulating CD27 is still unknown. The lack of somatic hypermutation in CVID seems to originate from the B cell side, since patients' T cells are able to induce somatic mutations in vitro in the Burkitt's lymphoma BL2 cells (27). Consistently, we never found an absence of or a decrease in CD70 expression on T cells, although it was observed by Brouet et al. (25). The fact that we followed the kinetics of CD70 expression over 5 days, whereas Brouet et al. made only one time-point measurement possibly missing the expression peak might explain these discrepancies. One could speculate that, in the subset of CVID identified in this study, a constitutive B cell defect impairs the T–B cross-talk leading to the acquisition of a memory B cell phenotype, including CD27 expression and Ig V gene somatic hypermutation. In this perspective, it is worth noting that B cells from certain CVID patients fail to up regulate CD86, a ligand for the T cell co-stimulatory molecule CD28, and CD137, another TNF receptor family member of unknown function on B cells (28). We are currently exploring the mechanisms regulating CD27 expression on B lymphocytes, whose identification will help characterize B cell abnormalities in CVID. In particular, it will be important to investigate these mechanisms in the subset of patients where no B cell CD27 expression is inducible after in vitro activation. Since these patients appear to have no circulating CD27+ B cells, it could be argued that the CD27 up-regulation obtained with the other patients results from the in vitro expansion of CD27+ B cells. We do not favor this interpretation, because the CD27 induction on CD27 separated B cells has been previously clearly established (8). One may also consider an alternative hypothesis for the reduced number of CD27+ B cells in CVID: CD27 might be normally induced on these patient B cells and trigger an abnormally high rate of apoptosis shrinking the CD27+ B cell pool. We have previously shown that CD27 cytoplasmic tail binds the protein Siva and that Siva has two isoforms, one of them, Siva-1, leading to apoptosis (29). The outcome of CD27 signaling requires a fine tuning between the differential binding of the two isoforms that might be ill balanced in CVID.

The second group of CVID patients that we and Brouet et al. (25) identified displays a normal number of CD27+ B cells. In these patients, B cell peripheral differentiation seems to be able to proceed one step further. Yet, some of them have an abnormal CD27 function, since in both series patients can be found whose B cells are unresponsive to the CD70/300-19 cells transfected with CD27 ligand. This finding suggests a defect in the as-yet uncharacterized CD27 signaling pathway leading to plasma cell differentiation. Looking at CD27, therefore, confirms the heterogeneous immunologic background of CVID since at least three patient profiles can be distinguished, i.e. low CD27 expression on B cells, normal expression with defective signaling and absence of CD27 abnormalities.

In total, our data provide evidence that a B cell defect in CD27 expression or signaling contributes to the altered plasma cell differentiation in subsets of CVID patients and shed some light on the importance of CD27 function in B cell biology.


    Acknowledgments
 
The authors thank Dr Prasad Kanteti for helpful discussions, Dr Paul Young and the nurses of the outpatient clinic for providing blood samples, and Miss Isabelle Duval for skillful secretarial assistance.


    Abbreviations
 
CD134L CD134 ligand
CVID common variable immunodeficiency
PBMC peripheral blood mononuclear cell
PE phycoerythrin
SAC Staphylococcus aureus Cowan I strain
TNF tumor necrosis factor

    Notes
 
Transmitting editor: S. Izui

Received 18 January 2001, accepted 28 March 2001.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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