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*
Department of Pediatrics, University of Washington, Seattle, WA 98195;
Department of Medicine III, Osaka University Medical School, Osaka, Japan;
Department of Pediatrics, Toyama Medical and Pharmaceutical University, Toyama, Japan; and
§
Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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X174 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
Sequence analysis of the Btk gene
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 analysis
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 x 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).
Stimulation of B cells with anti-CD40 mAb
To induce B cell proliferation, 2.5 x 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 x 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 x 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).
Immunization with bacteriophage X174
Bacteriophage X174 (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 x
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 x 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).
Statistics
Data were compared using the Student t test and StatView 4.01 software (Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1A). 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. 1B). Monocytes from mother of patient 5 showed a biphasic
pattern of Btk expression (21), indicating that the mother was a
carrier of XLA (Fig. 1B). 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.
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Proliferative responses of B cells
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).
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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.
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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).
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X174
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 X174. 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).
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X174, 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%). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 X174, 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 X174, 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shigeaki Nonoyama, Department of Pediatrics, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address:
3 Abbreviations used in this paper: XLA, X-linked agammaglobulinemia; xid, X-linked immunodeficiency; Kv, K value (rate of inactivation of bacteriophage X174); B-LCL, B lymphoblastoid cell line; Btk, Bruton tyrosine kinase; PE, phycoerythrin.
Received for publication April 2, 1998. Accepted for publication June 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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X174 in immunodeficiency diseases. J. Clin. Invest. 50:2559.
on CD40-mediated activation of B cells from X-linked immunodeficient or normal mice. J. Immunol. 159:1150.[Abstract]
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,
patient 2; 
, patient 3; 
, patient 4.