X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations of Bruton tyrosine kinase (Btk); Btk plays an essential role in the development of mature B cells. However, small numbers of B cells (“leaky B cells”) are present in the peripheral blood of most XLA patients. In this study, we analyzed the function of these leaky B cells obtained from XLA patients. Enough numbers of B cells were available for analysis from five of nine XLA patients originally screened. Sequence analysis revealed missense mutations of Btk in four of the five XLA patients. No mutation was found in the coding region of Btk in one patient. Western blotting and/or flow cytometric analysis failed to detect Btk protein in all five patients. B cells isolated from peripheral blood of these XLA patients were CD5, CD20+, CD19+, and CD21. If stimulated with anti-CD40 and IL-4, XLA B cells proliferated normally and produced significant amounts of IgE. Anti-CD40 stimulation of XLA B cells resulted in normal expression of CD23. In addition, three of the five XLA patients studied were immunized with bacteriophage φX174 and produced low but detectable levels of antiphage-specific Ab. Similarly, X-linked immunodeficiency mice, which carry a missense mutation in Btk, produced substantial amounts of antiphage Ab. These results indicate that CD40 signaling is intact in B cells lacking demonstrable Btk, and that leaky B cells in XLA patients can proliferate, undergo isotype switching, and differentiate into specific Ab-producing cells.

X-linked agammaglobulinemia (XLA)3 is a primary immunodeficiency disease characterized by severe hypogammaglobulinemia, defective B cell development, and severely decreased numbers of mature B cells. The gene responsible for XLA has been identified to be a cytoplasmic tyrosine kinase (Bruton tyrosine kinase (Btk)) (1, 2). To date, >200 different mutations have been reported in patients with XLA (3, 4, 5, 6). Btk is a Btk/Tec family cytoplasmic tyrosine kinase; it plays an essential role in the development and function of B cells. Lack of functional Btk, as observed in XLA patients, results in a block of the development of pro-B to pre-B cells and then to mature B cells.

However, most patients with XLA have detectable, although very few, B cells in the peripheral blood. Conley et al. reported that 38 of 44 patients had CD19+ B cells (between 0.01 and 1.0% of PBLs) (7). Jones et al. described two XLA patients who had 1 to 3% of CD19+ B cells (8). These “leaky” B cells in patients with XLA provide an excellent opportunity to examine the role of Btk in B cell function and differentiation. Studies of B cells obtained from X-linked immunodeficiency (xid) mice that have a naturally occurring missense mutation of Btk and of B cells from Btk knockout mice have demonstrated that Btk is involved in signaling pathways initiated by anti-Ig stimulation, anti-CD38 stimulation, IL-5, and IL-10 (9, 10, 11, 12, 13, 14, 15); alternatively, involvement of Btk in the CD40 signaling pathway is controversial (16, 17, 18). However, a careful functional analysis of B cells from XLA patients has not been conducted. Since xid mice and Btk-targeted mice present with a much milder phenotype than XLA patients, we analyzed the function of leaky B cells obtained from XLA patients to understand the role of Btk in human B cell development and function.

In this report, we demonstrate that the leaky B cells present in the circulation of patients with XLA can proliferate and produce IgE if stimulated with anti-CD40 and IL-4, similar to control B cells. In addition, in vivo immunization revealed that these B cells can produce low but detectable levels of specific Ab. These results indicate that leaky B cells in XLA patients are functional and suggest the presence of a compensatory pathway that can at least in part take over the role of Btk in human B cells.

Heparinized blood samples were obtained from healthy adult volunteers or patients with XLA. PBMCs were prepared by Ficoll-Hypaque gradient centrifugation. To further purify B cells, T cells were removed from mononuclear cells by twice rosetting with aminoethylisothiuronium bromide- (Sigma, St. Louis, MO) treated sheep RBCs (19, 20). NK cells and monocytes were removed by treating cells with 5 mM l-leucine methyl ester hydrochloride (Sigma) in serum-free RPMI 1640 as described previously (19, 20). In patients 4 and 5, B cells were further purified by Lympho-Kwik B (One Lambda, Canoga Park, CA). The resulting B cell-enriched populations obtained from XLA patients consisted of 50 to 60% B cells (CD20+) and <1% T cells (CD3+) as determined by flow cytometry. Substantial numbers of B cells for analysis were available from five of nine XLA patients originally screened. B cells obtained from five healthy adult volunteers were studied simultaneously and served as normal controls.

EBV-transformed B lymphoblastoid cell lines (B-LCLs) were derived from PBMCs infected with supernatants from the marmoset cell line B95–8. We were able to establish B-LCLs from four of the five XLA patients selected for the study.

Phenotypic analysis of freshly isolated B cells and B-LCLs was performed by flow cytometry using FITC- or phycoerythrin (PE)-conjugated anti-CD5, anti-CD19, anti-CD20, anti-CD21 (Coulter, Hialeah, FL), and anti-CD23 (Becton Dickinson, San Jose, CA).

Total RNA was isolated from PBMCs obtained from patients with XLA or from B-LCLs established from patients with XLA. First-strand cDNA was synthesized with the Superscript Preamplification System kit (Life Technologies, Gaithersburg, MD). PCR was performed using primer pairs covering the entire coding region of Btk cDNA as described previously (5). Direct sequencing was performed using a modified dideoxynucleotide chain termination method and the Pfu DNA-sequencing kit (Stratagene, La Jolla, CA). The mutations observed in the cDNA of patients were confirmed by sequencing genomic DNA.

Btk protein expression was determined either by Western blot analysis or by flow cytometry using anti-Btk mAb (48-2H) (4). For Western blot analysis, 1 × 107 B-LCLs from normal control subjects and patients with XLA were suspended in 1 ml of lysis buffer containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L PMSF, 0.5% aprotinin, and 10 μg/ml leupeptin at pH 7.5 and kept on ice for 10 min. Protein concentration was determined in each lysate using a protein assay kit (Bio-Rad, Hercules, CA). From each sample, 20 μg of total protein was loaded onto an SDS-polyacrylamide gel, electrophoresed, and transferred to a polyvinylidene difluoride Immobilon-P membrane (Millipore, Bedford, MA). After blocking with 10% nonfat milk, the membranes were incubated with anti-Btk mAb at 2 μg/ml or with anti-actin mAb (Sigma) at 1 μg/ml for 1 h at room temperature. After washing, membranes were incubated with alkaline phosphatase-conjugated anti-mouse IgG (Promega, Madison, WI) diluted 1/7500 for 1 h. Images were obtained with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer-Mannheim, Indianapolis, IN).

Flow cytometric analysis of intracytoplasmic Btk protein expression was performed as described previously (21). Briefly, PBMCs obtained from normal control subjects and patients with XLA were stained with PE-conjugated anti-CD14 mAb (Dako, Kyoto, Japan). Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washing, cells were reacted with anti-Btk mAb 48-2H for 20 min at 4°C and subsequently stained with FITC-conjugated anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL) for 20 min at 4°C. Expression of Btk protein in monocytes was analyzed by gating for CD14+ cells using a FACScan (Becton Dickinson).

To induce B cell proliferation, 2.5 × 104 freshly isolated B cells were cultured for 4 days in 96-well flat-bottom microculture plates at a final volume of 200 μl of RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), 2 mM glutamine, 50 U/ml penicillin, and 50 μg/ml of streptomycin (complete medium) with anti-CD40 mAb G28-5 (1 μg/ml) and IL-4 (100 U/ml) followed by a 16-h pulse with 1 μCi of [3H]thymidine. To induce CD23 expression, 10 × 104 purified B cells were cultured in 500 μl of complete medium in the presence of mAb G28-5 (1 μg/ml). After 16 h of culture, cells were dually stained with FITC-conjugated anti-CD20 mAb and PE-conjugated anti-CD23 mAb (Becton Dickinson). CD20+ B cells were examined for their CD23 expression by flow cytometry. To induce IgE production, 2.5 × 104 purified B cells were cultured in 96-well round-bottom microculture plates in 200 μl of complete medium in the presence of mAb G28-5 (1 μg/ml) and IL-4 (100 U/ml). After 12 days of culture, supernatants were collected and tested for IgE concentrations using an ELISA technique as described previously (19, 20).

Bacteriophage φX174 (phage), prepared as described previously (22, 23), was i.v. administered twice (6 wk apart) to three XLA patients (patients 2, 3, and 4) at the standard dose of 2 × 109 plaque-forming units/kg body weight. A total of 12 healthy, young, male volunteers were immunized similarly; sera were collected from both patients and controls immediately before immunization and at 1, 2, and 4 wk after each immunization.

Phage was given to mice i.v. at a dose of 2.5 × 108 plaque-forming units per mouse. CBA/N mice that carry the xid gene (xid mice) and xid congenic C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The primary immunization was followed 4 wk later by a secondary injection of the same amount of phage. Samples obtained by periorbital bleeding were collected immediately before immunization and at 1, 2, and 4 wk after each immunization.

Ab activity was determined by a sensitive phage neutralization assay and expressed as the rate of phage inactivation (K value, Kv) (22, 23). Neutralizing Ab resistant to 2-ME was considered to be IgG (22, 23).

Data were compared using the Student t test and StatView 4.01 software (Abacus Concepts, Berkeley, CA).

The five XLA patients selected for this study are from five unrelated families; each family has a history indicating x-linked inheritance. All patients had low or undetectable levels of serum IgG, IgA, and IgM, and the number of circulating CD20+ B cells was <1.0% as determined by flow cytometry (Table I). Western blot analysis revealed that B-LCLs established from four patients failed to express Btk (Fig. 1,A). FACS analysis was used to study patient 5, from whom a B-LCL could not be established; Btk protein was not detectable in the monocytes of this patient (Fig. 1,B). Monocytes from mother of patient 5 showed a biphasic pattern of Btk expression (21), indicating that the mother was a carrier of XLA (Fig. 1,B). These results demonstrated that all five patients lacked Btk protein expression. Sequence analysis of Btk revealed four different point mutations resulting in single amino acid substitutions (Table I). In patient 5, we were unable to identify a mutation within the coding region of the Btk gene.

Table I.

Laboratory findings of the XLA patients studied

PatientsAge (year)Ig Level (mg/dl)a
IgGIgAIgMB Cells (%)bBtk Protein ExpressionMutation of Btk
Pt. 1 80 <7 16 <1.0 — Arg28 → His 
Pt. 2 12 37 <7 <1.0 — Thr33 → Pro 
Pt. 3 15 344 <7 33 <1.0 — Leu408 → Pro 
Pt. 4 12 <7 17 <1.0 — Arg525 → Gln 
Pt. 5 24 177 <7 34 <1.0 — No mutation in the coding region 
PatientsAge (year)Ig Level (mg/dl)a
IgGIgAIgMB Cells (%)bBtk Protein ExpressionMutation of Btk
Pt. 1 80 <7 16 <1.0 — Arg28 → His 
Pt. 2 12 37 <7 <1.0 — Thr33 → Pro 
Pt. 3 15 344 <7 33 <1.0 — Leu408 → Pro 
Pt. 4 12 <7 17 <1.0 — Arg525 → Gln 
Pt. 5 24 177 <7 34 <1.0 — No mutation in the coding region 
a

Serum 1g concentrations were measured before IVIG therapy was introduced.

b

Percentages of CD20+ cells in peripheral blood are shown.

FIGURE 1.

Analysis of Btk protein expression in the XLA patients. A, Western blot analysis of Btk expression in B-LCLs established from four XLA patients (patients (Pts.) 1–4). Cell lysates were obtained from B-LCLs established from four XLA patients and one normal control. Btk and actin expression were analyzed by Western blot. B, Flow cytometric analysis of Btk expression in monocytes from an XLA patient. PBMCs obtained from an XLA patient (Pt. 5), his mother, and normal control were first stained with PE-conjugated anti-CD14 and then fixed, permeabilized, and stained with an anti-Btk mAb (48-2H) followed by FITC-conjugated anti-mouse Ig. Expression of Btk is shown as a solid line, and the negative control stained with mouse Ig followed by FITC-conjugated anti-mouse Ig is shown as a dashed line.

FIGURE 1.

Analysis of Btk protein expression in the XLA patients. A, Western blot analysis of Btk expression in B-LCLs established from four XLA patients (patients (Pts.) 1–4). Cell lysates were obtained from B-LCLs established from four XLA patients and one normal control. Btk and actin expression were analyzed by Western blot. B, Flow cytometric analysis of Btk expression in monocytes from an XLA patient. PBMCs obtained from an XLA patient (Pt. 5), his mother, and normal control were first stained with PE-conjugated anti-CD14 and then fixed, permeabilized, and stained with an anti-Btk mAb (48-2H) followed by FITC-conjugated anti-mouse Ig. Expression of Btk is shown as a solid line, and the negative control stained with mouse Ig followed by FITC-conjugated anti-mouse Ig is shown as a dashed line.

Close modal

Flow cytometric analysis of freshly isolated B cells from each of the five XLA patients revealed that the recovered B cells were CD19+, CD20+, CD21, CD23, and CD5 (data not shown). Similarly, B-LCLs established from four patients (patients 1–4) were CD19+, CD20+, CD21, and CD5, although they were uniformly positive for CD23 (data not shown).

B cell proliferation was studied by stimulating freshly isolated B cells with anti-CD40 and IL-4. Purified B cells from XLA patients and normal controls showed comparable [3H]thymidine uptake if cultured with anti-CD40 and IL-4 (Fig. 2). The stimulation index observed in normal B cells was 52.1 ± 29.2 (mean ± SD), and the index observed in XLA B cells was 60.3 ± 41.6 (p = 0.73, Student’s t test).

FIGURE 2.

Proliferative responses of B cells obtained from patients with XLA. Purified B cells obtained from patients with XLA and from age-matched healthy donors were cultured with or without anti-CD40 mAb (1 μg/ml) and IL-4 (100 U/ml) for 4 days followed by a 16-h pulse with 1 μCi of [3H]thymidine. Results are shown as stimulation indexes. Horizontal bars indicate geometric means of stimulation indexes.

FIGURE 2.

Proliferative responses of B cells obtained from patients with XLA. Purified B cells obtained from patients with XLA and from age-matched healthy donors were cultured with or without anti-CD40 mAb (1 μg/ml) and IL-4 (100 U/ml) for 4 days followed by a 16-h pulse with 1 μCi of [3H]thymidine. Results are shown as stimulation indexes. Horizontal bars indicate geometric means of stimulation indexes.

Close modal

B cells obtained from three XLA patients (patients 1, 2, and 5) and from normal subjects were stimulated with anti-CD40 for 16 h, and the expression of CD23 was examined by flow cytometry. B cells from XLA patients expressed anti-CD40-induced CD23 at concentrations comparable with those of normal B cells (patient 1, 18.2%; patient 2, 14.0%; patient 5, 17.7%; and controls, 21.7%). Typical results observed in patient 1 and in a normal control are shown in Figure 3.

FIGURE 3.

Induction of CD23 in B cells obtained from patients with XLA. Purified B cells obtained from patients with XLA and from age-matched healthy donors were cultured in the absence (a and c) or presence (b and d) of anti-CD40 mAb (1 μg/ml) for 16 h and dually stained with FITC-conjugated anti-CD20 and PE-conjugated anti-CD23. CD20+ B cells were gated and examined for their CD23 expression (shaded curve) by flow cytometry. PE-conjugated mouse Ig (open curve) was used as a negative control.

FIGURE 3.

Induction of CD23 in B cells obtained from patients with XLA. Purified B cells obtained from patients with XLA and from age-matched healthy donors were cultured in the absence (a and c) or presence (b and d) of anti-CD40 mAb (1 μg/ml) for 16 h and dually stained with FITC-conjugated anti-CD20 and PE-conjugated anti-CD23. CD20+ B cells were gated and examined for their CD23 expression (shaded curve) by flow cytometry. PE-conjugated mouse Ig (open curve) was used as a negative control.

Close modal

As shown in Figure 4, purified B cells from all normal subjects (n = 5) produced IgE if cultured for 12 days in the presence of anti-CD40 mAb and IL-4. Mean ± SD of IgE was 16,107 ± 5,096 pg/ml. Using the same culture system, XLA B cells were studied for IgE production (Fig. 4). Although the amount of IgE produced varied, B cells from all five XLA patients produced IgE at a concentration of 13,795 ± 9,429 pg/ml (mean ± SD), which is not significantly different from normal controls (p = 0.75, Student’s t test). Without stimulation, B cells from XLA patients failed to produce detectable levels of IgE (<200 pg/ml).

FIGURE 4.

IgE production by B cells obtained from patients with XLA. Purified B cells obtained from patients with XLA and from age-matched healthy donors were cultured with or without anti-CD40 mAb (1 μg/ml) and IL-4 (100 U/ml) for 12 days. IgE concentrations in culture supernatants were analyzed by ELISA. −, cultured without anti-CD40 and IL-4; +, cultured with anti-CD40 and IL-4. Horizontal bars indicate geometric means of the IgE concentrations produced.

FIGURE 4.

IgE production by B cells obtained from patients with XLA. Purified B cells obtained from patients with XLA and from age-matched healthy donors were cultured with or without anti-CD40 mAb (1 μg/ml) and IL-4 (100 U/ml) for 12 days. IgE concentrations in culture supernatants were analyzed by ELISA. −, cultured without anti-CD40 and IL-4; +, cultured with anti-CD40 and IL-4. Horizontal bars indicate geometric means of the IgE concentrations produced.

Close modal

To measure in vivo Ab responses to a T-dependent neo-antigen, we immunized 3 of the 5 XLA patients (patients 2, 3, and 4) and 12 normal controls with bacteriophage φX174. As shown in Figure 5, normal controls show a characteristic pattern of neutralizing Ab production, including amplification of titers and switch from IgM to IgG (% IgG = 47%) after a secondary immunization. The XLA patients produced very low but detectable amounts of antiphage Ab, although isotype switching was not observed (% IgG = 0%) after a secondary immunization (Fig. 5).

FIGURE 5.

In vivo production of phage-neutralizing Ab by normal humans and patients with XLA. Normal humans (n = 12) and three patients with XLA were immunized twice with bacteriophage φX174. Sera were collected at the time indicated, and antiphage Ab titers (Kv) were measured by a neutralizing assay. •, geometric means of controls; dotted line, ±1 SD of controls; ○, patient 2; ▵, patient 3; □, patient 4.

FIGURE 5.

In vivo production of phage-neutralizing Ab by normal humans and patients with XLA. Normal humans (n = 12) and three patients with XLA were immunized twice with bacteriophage φX174. Sera were collected at the time indicated, and antiphage Ab titers (Kv) were measured by a neutralizing assay. •, geometric means of controls; dotted line, ±1 SD of controls; ○, patient 2; ▵, patient 3; □, patient 4.

Close modal

Following immunization with bacteriophage φX174, xid mice produced substantial amounts of antiphage Ab, although the Ab titers produced by xid mice were significantly lower than those produced by control mice. The peak Kv (mean ± SD) after primary immunization was 0.859 ± 0.301 in xid mice and 6.595 ± 1.979 in control mice (p < 0.01, Student’s t test); the peak Kv after secondary immunization was 147.9 ± 95.9 in xid mice and 406.7 ± 165.1 in control mice (p < 0.01, Student’s t test). The isotype switching observed after a secondary immunization in xid mice (% IgG = 64%) was comparable with that observed in normal congenic mice (% IgG = 79%).

Patients with XLA due to a mutation of Btk may present with variable clinical phenotypes and laboratory findings (24). Although a very low number of circulating B lymphocytes is a hallmark of XLA, a B cell number in the peripheral blood of >0.5% is often associated with a milder phenotype and with the production of limited quantities of specific Ab following immunization (24). Using standard techniques, we were able to isolate sufficient B lymphocytes to perform functional analysis from the peripheral blood of five of nine XLA patients available for study. When stimulated with anti-CD40 mAb and IL-4, B lymphocytes from XLA patients proliferated normally and produced near normal quantities of IgE, although enrichment of B cells from the blood of XLA patients resulted in a cell population consisting of only 50 to 60% CD20+ B cells as compared with >90% CD20+ B cells isolated from control blood. Furthermore, CD23 expression by XLA B lymphocytes following stimulation with anti-CD40 mAb was comparable with that of normal control B cells. The demonstration of absent Btk expression in EBV-induced lymphoblasts and in monocytes from all five of the XLA patients studied suggests that signaling through CD40 occurs normally in patient B cells despite the absence of Btk. In contrast, B cells isolated from xid mice do not proliferate (17) and fail to up-regulate CD80 and CD86 expression (18) following ligation of CD40, suggesting that CD40 signaling is defective in xid mice with the Arg28 → Cys mutation of Btk. One possible explanation for this discrepancy is a difference in the potency of CD40 stimulation between the anti-CD40 mAb used in xid mice and the anti-CD40 mAb that we used in the human system. The recent observation that xid B cells proliferate normally if stimulated with soluble CD40 ligand (16) or with a different anti-CD40 mAb further supports this possibility (25). Obviously, the precise role of Btk in CD40 signaling pathways in both mice and humans requires further investigation.

Three of the five XLA patients studied were immunized with bacteriophage φX174, a T-dependent neo-antigen; in normal controls, this neo-antigen induces a classic Ab response consisting of immunologic memory, amplification of Ab titers, and switch from IgM to IgG following secondary exposure to the Ag. Despite absent Btk expression, each of the three patients produced detectable amounts of phage-neutralizing Ab, although at low titers and minimal amplification and without switching. Others have observed a subgroup of XLA patients with demonstrated Btk mutations that was able to produce specific Ab to diphtheria and tetanus toxoid, poliomyelitis, and influenza immunization (8, 26). xid mice showed an even stronger response to bacteriophage φX174, including amplification and switch from IgM to IgG, and responded almost normally to trinitrophenyl-LPS, a T-independent Ag, and trinitrophenyl-keyhole limpet hemocyanin, a T-dependent Ag (10). These observations indicate that B cells can differentiate into specific Ab-producing lymphocytes despite our inability to demonstrate Btk protein. However, ∼40% of a group of 36 XLA patients immunized with bacteriophage failed to clear Ag and produce Ab. Those XLA patients that were able to generate phage-specific Ab did so at very low titers and failed to switch from IgM to IgG (24). This pattern of in vivo Ab responses may reflect the generation of a very limited number of B cell clones in some XLA patients or the complete absence of Ag-specific B cell clones in others. It is of interest that the four XLA patients that we had to exclude from this study due to insufficient B cell numbers in the peripheral blood failed to clear phage and produce detectable antiphage Ab. The formidable, although depressed, response of xid mice to T-dependent Ags may be related to the relatively high B cell number in xid mice (50% of normal mice).

The circulating B cells obtained from the five XLA patients studied resemble conventional B cells. They were found to have the normal phenotype CD19+, CD20+, and CD5, similar to circulating B cells in xid mice that also have a conventional phenotype of B cells (10). It is tempting to speculate that the leaky B cells observed in XLA patients have differentiated using Btk-independent pathways. A role of CD40 in this differentiation process is suggested by the finding of Oka et al., who reported that xid mice that were made simultaneously CD40 showed a profound reduction of mature B cells (27). These authors hypothesize that mature B cells are usually generated through a Btk-dependent pathway, but, alternatively, may also be generated through a CD40-controlled pathway. Based on this hypothesis, the cells observed in xid mice would have been generated by a CD40-controlled pathway. Mice that were targeted for both Btk and CD40 gene deletions had a more severely impaired B cell maturation than xid mice (28). Therefore, it is possible that the circulating B cells observed in XLA patients are derived from CD40-controlled, Btk-independent pathways. This hypothesis is consistent with our observation that CD40 signaling is intact in XLA B cells. The difference between the severely depressed B cell numbers observed in XLA patients and the moderately reduced B cell numbers characteristic for xid- or Btk-targeted mice suggests that the CD40-controlled pathway is insufficient for effective B cell differentiation in humans. Alternatively, it is possible that the leaky B cells obtained from XLA patients with missense mutations of Btk have a minimal amount of functional Btk that is not demonstrable with flow cytometry or Western blot analysis. The fact that XLA B cells proliferate normally and undergo isotype switching in vitro supports a novel treatment strategy that involves expanding leaky B cells in vitro by CD40 stimulation that could be injected into the patient. Such a therapy could be of use for a subgroup of XLA patients.

We thank Dr. Shigeyuki Arai (Fujisaki Institute, Hayashibara Biochemical Laboratories Inc., Okayama, Japan) for help in generating anti-Btk mAb 48-2H.

1

This work was supported in part by grants from the National Institutes of Health (HD17427) (to H.D.O.), the March of Dimes and Birth Defects Foundation (G-0116) (to H.D.O.), the Uehara Memorial Foundation, the Ryoichi Naito Foundation for Medical Research, and the Ministry of Education, Science, and Culture (SN 08282214).

3

Abbreviations used in this paper: XLA, X-linked agammaglobulinemia; xid, X-linked immunodeficiency; Kv, K value (rate of inactivation of bacteriophage φX174); B-LCL, B lymphoblastoid cell line; Btk, Bruton tyrosine kinase; PE, phycoerythrin.

1
Tsukada, S., D. C. Saffran, D. J. Rawlings, O. Parolini, R. C. Allen, I. Klisak, R. S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, J. W. Belmont, M. D. Cooper, M. E. Conley, O. N. Witte.
1993
. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.
Cell
72
:
279
2
Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow, C. I. E. Smith, D. R. Bentley.
1993
. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.
Nature
361
:
226
3
Vihinen, M., T. Iwata, C. Kinnon, S. P. Kwan, H. D. Ochs, I. Vorechovsky, C. I. E. Smith.
1996
. BTKbase, mutation database for X-linked agammaglobulinemia (XLA).
Nucleic Acids Res.
24
:
160
4
Hashimoto, S., Tsukada S., Matsushita M., Miyawaki T., Niida Y., Yachie A., Kobayashi S., Iwata T., Hayakawa H., Matsuoka H., Tsuge I., Yamadori T., Kunikata T., Arai S., Yoshizaki K., et al
1996
. Identification of Bruton’s tyrosine kinase (Btk) gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: a nationwide study of Btk deficiency in Japan.
Blood
88
:
561
5
Zhu, Q., M. Zhang, J. Winkelstein, S. H. Chen, H. D. Ochs.
1994
. Unique mutations of Bruton’s tyrosine kinase in fourteen unrelated X-linked agammaglobulinemia families.
Hum. Mol. Genet.
3
:
1899
6
Vihinen, M..
1996
. BTKbase: XLA-mutation registry.
Immunol. Today
17
:
502
7
Conley, M. E..
1985
. B cells in patients with X-linked agammaglobulinemia.
J. Immunol.
134
:
3070
8
Jones, A., L. Bradley, L. Alterman, M. Tarlow, R. Thompson, C. Kinnon, G. Morgan.
1996
. X-linked agammaglobulinaemia with a “leaky” phenotype.
Arch. Dis. Child.
74
:
548
9
Rawlings, D. J., D. C. Saffran, S. Tsukada, D. A. Largaespada, J. C. Grimaldi, L. Cohen, R. N. Mohr, J. F. Bazan, M. Howard, N. G. Copeland, N. A. Jenkins, O. N. Witte.
1993
. Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient xid mice.
Science
261
:
358
10
Wicker, L. S., I. Scher.
1986
. X-linked immune deficiency (xid) of CBA/N mice.
Curr. Top. Microbiol. Immunol.
124
:
87
11
Hitoshi, Y., E. Sonoda, Y. Kikuchi, S. Yonehara, H. Nakauchi, K. Takatsu.
1993
. IL-5 receptor-positive B cells, but not eosinophils, are functionally and numerically influenced in mice carrying the X-linked immune defect.
Int. Immunol.
5
:
1183
12
Santos-Argumedo, L., F. E. Lund, A. W. Heath, N. Solvason, W. W. Wu, J. C. Grimaldi, R. M. Parkhouse, M. Howard.
1995
. CD38 unresponsiveness of xid B cells implicates Bruton’s tyrosine kinase (btk) as a regulator of CD38-induced signal transduction.
Int. Immunol.
7
:
163
13
Go, N. F., B. E. Castle, R. Barrett, R. Kastelein, W. Dang, T. R. Mosmann, K. W. Moore, M. Howard.
1990
. Interleukin 10, a novel B cell stimulatory factor: unresponsiveness of X chromosome-linked immunodeficiency B cells.
J. Exp. Med.
172
:
1625
14
Kerner, J. D., M. W. Appleby, R. N. Mohr, S. Chien, D. J. Rawlings, C. R. Maliszewski, O. N. Witte, R. M. Perlmutter.
1995
. Impaired expansion of mouse B cell progenitors lacking Btk.
Immunity
3
:
301
15
Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Muller, A. B. Kantor, L. A. Herzenberg, F. S. Rosen, P. Sideras.
1995
. Defective B cell development and function in Btk-deficient mice.
Immunity
3
:
283
16
Anderson, J. S., M. Teutsch, Z. Dong, H. H. Wortis.
1996
. An essential role for Bruton’s tyrosine kinase in the regulation of B-cell apoptosis.
Proc. Natl. Acad. Sci. USA
93
:
10966
17
Hasbold, J., G. G. Klaus.
1994
. B cells from CBA/N mice do not proliferate following ligation of CD40.
Eur. J. Immunol.
24
:
152
18
Goldstein, M. D., M. A. Debenedette, D. Hollenbaugh, T. H. Watts.
1996
. Induction of costimulatory molecules B7-1 and B7-2 in murine B cells: the CBA/N mouse reveals a role for Bruton’s tyrosine kinase in CD40-mediated B7 induction.
Mol. Immunol.
33
:
541
19
Nonoyama, S., M. L. Farrington, H. Ishida, M. Howard, H. D. Ochs.
1993
. Activated B cells from patients with common variable immunodeficiency proliferate and synthesize immunoglobulin.
J. Clin. Invest.
92
:
1282
20
Nonoyama, S., L. A. Penix, C. P. Edwards, D. B. Lewis, S. Ito, A. Aruffo, C. B. Wilson, H. D. Ochs.
1995
. Diminished expression of CD40 ligand by activated neonatal T cells.
J. Clin. Invest.
95
:
66
21
Futatani, T., T. Miyawaki, S. Tsukada, S. Hashimoto, T. Kunikata, S. Arai, M. Kurimoto, Y. Niida, H. Matsuoka, Y. Sakiyama, T. Iwata, S. Tsuchiya, O. Tatsuzawa, K. Yoshizaki, T. Kishimoto.
1998
. Deficient expression of Bruton’s tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection.
Blood
91
:
595
22
Nonoyama, S., D. Hollenbaugh, A. Aruffo, J. A. Ledbetter, H. D. Ochs.
1993
. B cell activation via CD40 is required for specific Ab production by Ag-stimulated human B cells.
J. Exp. Med.
178
:
1097
23
Ochs, H.D., S. D. Davis, R. J. Wedgwood.
1971
. Immunologic responses to bacteriophage φX174 in immunodeficiency diseases.
J. Clin. Invest.
50
:
2559
24
Ochs, H. D., C. I. E. Smith.
1996
. X-linked agammaglobulinemia: a clinical and molecular analysis.
Medicine
75
:
287
25
Johnson-Leger, C., J. Hasbold, M. Holman, G. G. Klaus.
1997
. The effects of IFN-γ on CD40-mediated activation of B cells from X-linked immunodeficient or normal mice.
J. Immunol.
159
:
1150
26
De Weers, M., G. M. Dingjan, G. S. Brouns, M. E. M. Kraakman, R. G. J. Mensink, R. C. Lovering, R. K. B. Schuurman, J. Borst, R. W. Hendriks.
1997
. Expression of Bruton’s tyrosine kinase in B lymphoblastoid cell lines from X-linked agammaglobulinaemia patients.
Clin. Exp. Immunol.
107
:
235
27
Oka, Y., A. G. Rolink, J. Andersson, M. Kamanaka, J. Uchida, T. Yasui, T. Kishimoto, H. Kikutani, F. Melchers.
1996
. Profound reduction of mature B cell numbers, reactivities, and serum Ig levels in mice which simultaneously carry the xid and CD40 deficiency genes.
Int. Immunol.
8
:
1675
28
Khan, W. N., A. Nilsson, E. Mizoguchi, E. Castigli, J. Forsell, A.K. Bhan, R. Geha, P. Sideras, F. W. Alt.
1997
. Impaired B cell maturation in mice lacking Bruton’s tyrosine kinase (Btk) and CD40.
Int. Immunol.
9
:
395